Chemical Constituents of Lecythispisonis (Lecythidaceae)--A New Saponin and Complete 1H and 13C Chemical Shift Assignments.

A novel triterpenoid saponin 3-O-ß-D-glucuronopyranosyl-(1' --> 3)-2α,19α-dihydroxyolean-12-en-28-oic acid [3-O-ß-D-glucuronopyranosyl-(1' --> 3)-arjunic acid, 1], ten known compounds [six triterpenoids: α-amyrin (2), ß-amyrin (3), germanicol (4), lupeol (5), friedelin (6), friedelanol (7); four steroids--campesterol (8), stigmasterol (9), sitosterol (10), cholesterol (11)], and a long chain alcohol n-eicosan-1-ol (12) were identified in the bark of Lecythis pisonis. The structures were established by 1D and 2D NMR spectroscopy (1H and 13C-NMR, DEPTQ, 1H-1H-COSY, NOESY, HSQC and HMBC), low (CG-MS) and high resolution mass spectrometry (HR-ESI-MS), and infrared (IR) spectral data involving comparison with the literature.

[On the reliability of brainstem electric response audiometry (BERA)]. / Zur Verlässlichkeit hirnstammaudiometrischer Hörschwellenbestimmungen.

BACKGROUND: Brainstem electric response audiometries (BERA) are in clinical use for a number of years. The aim of our study was to evaluate data regarding the long-term reliability of BERA-determined frequency specific thresholds in hearing disabled children. MATERIAL AND METHODS: In a group of 97 hearing disabled children we sought to compare Notched-Noise- (NN) BERA threshold as well as Click-BERA thresholds taken shortly after birth with behavioral audiometry thresholds determined after 3.2 years (mean). RESULTS: We found a significant correlation between both BERA methods and the behavioral tests. However, the correlation coefficients for NN-BERA were higher than for Click-BERA thresholds. CONCLUSION: Our results provide evidence for a high reliability of the NN-BERA for characterization of early onset hearing disabilities in children. Our data suggest that pathologic findings in the Click-BERA should always be followed by a frequency specific analysis with NN-BERA.

[Gross haematuria as the primary symptom of extramedullary IgM plasmacytoma of the bladder]. / Makrohämaturie als Primärsymptom eines extramedullären IgM-Plasmozytoms der Harnblase.

Haematuria is the most common clinical symptom of bladder cancer. Besides antibiotic treatment of a probably existing urinary tract infection, ultrasonography of the urinary organs, diagnostic cystoscopy (with biopsy if needed), and radiologic evaluation of the upper urinary tract (intravenous urography, computed tomography or magnetic resonance urography, retrograde pyelography) should be done for further evaluation. Atypical manifestations of systemic diseases with bladder infiltration could feign the clinical appearance of chronic cystitis and hinder determination of the correct diagnosis. The case of a 40-year-old man with recurrent gross haematuria due to extremely rare bladder infiltration through an IgM plasmacytoma is presented.

[Receptor tyrosine kinases in Hodgkin lymphoma as possible therapeutic targets]. / Rezeptor-Tyrosinkinasen in Hodgkin-Lymphomen als mögliche Angriffspunkte neuer Therapieoptionen.

Hodgkin lymphoma (HL) is the most frequent nodal lymphoma in Europe. The B-cell derived Hodgkin-Reed Sternberg (HRS) cells are nearly completely deficient for expression of B-cell markers. Epstein-Barr virus (EBV) can be detected in about 40% of HL cases. Presumably, EBV protects HRS cell precursors from apoptosis. Histologically only single HRS cells are dispersed in a broad reactive cellular background. Interactions between HRS cells and their surrounding cellular infiltrate, among them paracrine activation of several signalling pathways, is crucial in HL. HRS cells also show autocrine activation of several signalling pathways. Among these, the aberrant expression and activation of seven different receptor tyrosine kinases (RTK) is of special interest, as many different antibodies and low molecular substances which inhibit RTK activity are already in clinical use for anticancer therapy. Therefore, blocking of RTK activities in HL may be a novel therapeutic option.

High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma.

Mediastinal large B-cell (MBL) and classical Hodgkin lymphoma (HL) have several pathogenic mechanisms in common. As we recently observed aberrant tyrosine kinase (TK) activities in HL, we now analysed also MBL for such activities. Indeed, MBL and HL were the only B-cell lymphomas where elevated cellular phospho-tyrosine contents were typical features. Three TKs, JAK2, RON and TIE1, not expressed in normal B cells, were each expressed in about 30% of MBL cases, and 75% of cases expressed at least one of the TKs. Among the intracellular pathways frequently triggered by TKs, the PI3K/AKT pathway was activated in about 40% of MBLs and essential for survival of MBL cell lines, whereas the RAF/mitogen-activated protein kinase pathway seemed to be inhibited. No activating mutations were detected in the three TKs in MBL cell lines and primary cases. RON and TIE1 were each also expressed in about 35% and JAK2 in about 53% of HL cases. JAK2 genomic gains are frequent in MBL and HL but we observed no strict correlation of JAK2 genomic status with JAK2 protein expression. In conclusion, aberrant TK activities are a further shared pathogenic mechanism of MBL and HL and may be interesting targets for therapeutic intervention.

Structure of the light chain-binding domain of myosin V.

Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of approximately 36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-A resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2-IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM-IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein-protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V.

Insights into the multistep transformation process of lymphomas: IgH-associated translocations and tumor suppressor gene mutations in clonally related composite Hodgkin's and non-Hodgkin's lymphomas.

Clonally related composite lymphomas of Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL) represent models to study the multistep transformation process in tumorigenesis and the development of two distinct tumors from a shared precursor. We analyzed six such lymphomas for transforming events. The HLs were combined in two cases with follicular lymphoma (FL), and in one case each with B-cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). In the HL/FL and HL/MCL combinations, BCL2/IGH and CCND1/IGH translocations, respectively, were detected in both the HL and NHL. No mutations were found in the tumor suppressor genes FAS, NFKBIA and ATM. The HL/DLBCL case harbored clonal replacement mutations of the TP53 gene on both alleles exclusively in the DLBCL. In conclusion, we present the first examples of molecularly verified IgH-associated translocations in HL, which also show that BCL2/IGH or CCND1/IGH translocations can represent early steps in the pathogenesis of composite HL/FL or HL/MCL. The restriction of the TP53 mutations to the DLBCL in the HL/DLBCL case exemplifies a late transforming event that presumably happened in the germinal center and affected the fate of a common lymphoma precursor cell towards development of a DLBCL.

Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs-functional implications.

IQ motifs are widespread in nature. Mlc1p is a calmodulin-like myosin light chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP-related protein, Iqg1p, playing a role in polarized growth and cytokinesis in Saccharomyces cerevisiae. The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically. When bound to IQ2, Mlc1p adopts a compact conformation in which both the N- and C-lobes interact with the IQ motif. However, in the complex with IQ4, the N-lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p. The two light chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N-lobes of calmodulin-like light chains. The correlation between light chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p. The resulting 'free' N-lobes of myosin light chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment.

Crystallization, X-ray characterization and selenomethionine phasing of Mlc1p bound to IQ motifs from myosin V.

Mlc1p is a calmodulin-like protein from the budding yeast Saccharomyces cerevisiae, where it has been identified as a subunit of a class V myosin, Myo2p, and a binding partner of an IQGAP-like protein, Iqg1p. Through its interactions with these two proteins, Mlc1p plays a role in polarized growth and cytokinesis. Mlc1p has been crystallized in complexes with four different IQ target motifs from the neck region of Myo2p: IQ2, IQ3, IQ4 and IQ2-IQ3 (referred to as IQ2,3). Electron-density maps for two of the complexes (Mlc1p-IQ4 and Mlc1p-IQ2,3) were obtained from multiple anomalous dispersion (MAD) experiments based on selenomethionine derivatives. The other two structures (Mlc1p-IQ2 and Mlc1p-IQ3) were determined by molecular replacement using the partially refined structure of Mlc1p-IQ2,3 as a search model.

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6.

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.

Immunoadhesins of interleukin-6 and the IL-6/soluble IL-6R fusion protein hyper-IL-6.

Signal transduction in response to interleukin-6 (IL-6) results from homodimerization of gp130. This dimerization occurs after binding of IL-6 to its surface receptor (IL-6R) and can also be triggered by the complex of soluble IL-6R and IL-6. We fused IL-6 to the constant region of a human IgG1 heavy chain (Fc). IL-6Fc was expressed in COS-7 cells and purified via Protein A Sepharose. Using three different assays we found that the biological activity of this dimeric IL-6 protein is comparable with monomeric IL-6. Recently, we described the designer cytokine Hyper-IL-6 (H-IL-6) in which soluble IL-6R and IL-6 are connected via a flexible peptide linker. This molecule turned out to be 100-1000 times more effective than unlinked IL-6 and soluble IL-6R. Hyper-IL-6 acts on cells only expressing gp130 and is a potent stimulator of in vitro expansion of early hematopoietic precursors. Here we show that a Fc fusion protein of H-IL-6 (H-IL-6Fc) has the same biological activity on BAF/gp130 cells as H-IL-6. Furthermore, both H-IL-6 forms have a similar ability to induce the synthesis of acute phase proteins in human hepatoma cells HepG2 and in mice in vivo. The introduction of a thrombin cleavage site between H-IL-6 and the Fc portion of H-IL-6Fc made it possible to specifically recover biologically active monomeric H-IL-6 by limited proteolysis of the fusion protein. A more general use of cleavable immunoadhesins expressed in mammalian cells is discussed.

A new type of cytokine receptor antagonist directly targeting gp130.

The interleukin-6-type family of cytokines bind to receptor complexes that share gp130 as a common signal-transducing subunit. So far, receptor antagonists for interleukin-6-type cytokines have been constructed that still bind to the specific ligand binding subunit of the receptor complex, but have lost the ability to stimulate gp130. Such receptor antagonists compete for a specific receptor of a member of the cytokine family. Interleukin-6 only binds to gp130 when complexed with the interleukin-6 receptor that exists as a membrane bound and soluble molecule. Here we have constructed fusion proteins that consist of the soluble form of the human interleukin-6 receptor covalently linked to interleukin-6 receptor antagonists. These fusion proteins directly bind to gp130. Moreover, at concentrations of 10-50 nM they completely neutralize not only the biological activity of interleukin-6 but also of other cytokines of the interleukin-6-type family that act via gp130 homodimers or gp130/LIF-R heterodimers. Therefore, these gp130 targeting cytokine antagonists might be useful therapeutic tools in disease states that are related to cytokines of the interleukin-6 family.

Training children with asthma to use inhalation therapy equipment.

In Experiment I, four children with asthma were taught to use the intermittent positive-pressure breathing (IPPB) apparatus, a device that delivers bronchodilator medication to the lungs under positive pressure. Because these youngsters had not learned to use the device with repeated instructions, script with back-up reinforcement was introduced to train sequentially three responses - eye fixation, facial posturing, and diaphragmatic breathing - according to a multiple-baseline design. The procedures were effective in teaching appropriate use of the IPPB apparatus. Further, the children's use of the apparatus after training resulted in significantly more effective relief of asthma symptoms. In a second experiment, nurses were instructed in the application of the operant techniques used in the first study, and then served as experimenters in a partial replication of Experiment I. The data once again reflected a strong impact of the intervention program on IPPB responses.