The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO 163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO 163 (1) predicted the existence of a potent, lineage-specific soluble factor, which they called thrombopoietin (TPO), that stimulates megakaryocytopoiesis and platelet production. It was not until 1994 that unequivocal evidence for the existence of this elusive molecule was provided by the nearly simultaneous isolation and cloning of TPO by five independent research groups (2-6). This cytokine has proven to be a primary factor in megakaryocytopoiesis from megakaryocyte colony formation to platelet production and the differentiation and proliferation of progenitor cells of multiple hematopoietic lineages (7). As such, TPO is being investigated for its potential to treat thrombocytopenia resulting from AIDS and chemotherapy and radiation treatments for cancer and leukemia and for the in vivo and ex vivo expansion of hematopoietic stem cells for bone marrow transplantation.Human TPO (hTPO) is a heavily glycosylated protein with two distinct regions. The 153-residue N-terminal region is homologous to human erythropoietin (EPO) with which it shares 23% sequence identity and is sufficient for receptor binding and signal transduction (2,3,8). The 179-residue C-terminal region has a large number of proline and glycine residues and six N-linked glycosylation sites. Its function is not known, although recent work indicates a role in secretion and protection from proteolysis (9, 10).The TPO receptor c-Mpl was first identified as an oncogene of the murine myeloproliferative leukemia virus (11, 12) that was able to immortalize hematopoietic progenitor cells and was later cloned from human and mouse (13,14). c-Mpl is expressed in some pluripotent hematopoietic stem cells (15) and in the megakaryocyte lineage from progenitor cells to platelets (16). It is a class I cytokine receptor of the hematopoietic superfamily of receptors and signals by the JAK͞STAT, Ras, and mitogenactivated protein kinase pathways (17-21). Class I hematopoietic receptors bind to their cytokine ligands by Ϸ200-aa Ig-like extracellular domains called cytokine receptor homology (CRH) or hematopoietic receptor domains that contain a distinctive WSXWS sequence motif (13).Cytokines possess two distinct interaction sites that bind with differing affinities [high affinity (nanomolar range) and low affinity (micromolar range)] to the same cytokinerecognition surface of the CRH domain. Crystal structures of human EPO and human growth hormone (hGH) in complex with the extracellular CRH domains of their receptors (22, 23) have shown the cytokine-CRH interaction in detail. However, unlike EPO receptor (EPOR) and hGH receptor (hGHR), which have only one CRH domain, c-Mpl belongs to a subset of hematopoietic ...

α-Acids and β-acids, two main components of hop resin, are known to be susceptible to oxygen and degraded during hop storage, although the oxidation products in stored hops have not been fully identified. In this study, we developed a high-performance liquid chromatography (HPLC) analysis method suitable for separation and quantification of the oxidation products. This HPLC analysis clearly proved, for the first time, that humulinones and hulupones are major products in oxidized hops. We are also the first to identify novel 4'-hydroxy-allohumulinones, suggested to be oxidative products of humulinones, by means of NMR spectroscopy and high-resolution mass spectrometry. Using the developed analytical method, changes in α- and β-acids and their oxidation products during hop storage were clearly revealed for the first time.

The bitter acids in hops (Humulus lupulus L.) and beer, such as α-, β-, and iso-α-acids, are known to affect beer quality and display various physiological effects. However, these compounds readily oxidize, and the effect of the oxides on the properties of beer or their potential health benefits are not well understood. In this study, we developed a simple preparative method for the bitter acid oxide fraction derived from hops and designated the constituents as matured hop bitter acids (MHBA). HPLC-PDA-ESI/HRMS and MS(2) revealed that MHBA are primarily composed of α-acid-derived oxides, which possess a common β-tricarbonyl moiety in their structures similar to α-, β-, and iso-α-acids. We also developed a quantitative analytical method of whole MHBA by HPLC, which showed high precision and reproducibility. Using our newly developed method, the concentration of whole MHBA in several commercial beers was evaluated. Our results will promote the study of bitter acid oxides.

The resins from hops (Humulus lupulus L.), which add the bitter taste to beer, are classified into two main sub-fractions, namely, soft and hard resins. α- and β-Acids in soft resin and their transformation during the wort boiling process are well-studied; however, other constituents in resins, especially hard resin, have been unidentified. In this study, we identified humulinones and hulupones as soft-resin components, in addition to 4'-hydroxyallohumulinones and tricyclooxyisohumulones A and B as hard-resin components. These compounds are all oxidation products derived from α- or β-acids. We also investigated compositional changes in the hard resin during the wort boiling process, which has a significant effect on the taste of the beer, by using model boiling experiments. The major changes were identified to be isomerization of 4'-hydroxyallohumulinones into 4'-hydroxyallo-cis-humulinones, followed by decomposition into cis-oxyhumulinic acids. These findings will be helpful in systematically evaluating and optimizing the effect of the hard resin on beer quality.

The transformation of α-acids [in hops (Humulus lupulus L.)] to iso-α-acids (in beer) during the brewing process is well known, but the occurrence and structure of the oxidized α-acids during hop storage are not well documented. Because an understanding of these oxidized compounds is essential to optimize the effects of oxidized hops on the quality of beer, we investigated the autoxidation products of humulone (a representative congener of α-acids) using a simplified autoxidation model. Among the oxidation products, tricyclooxyisohumulones A (1) and B (2), tricycloperoxyisohumulone A (3), deisopropyltricycloisohumulone (4), and the hemiacetal 5 of tricycloperoxyhumulone A (5') were isolated, and their structures were elucidated for the first time. The occurrence of compounds 1-4 in stored hops was verified using LC/MS/MS analysis. We also monitored the levels of compounds 1-4 during hop storage using LC/MS/MS analysis.