A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B-and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8-T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the manor cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous nijection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 iniection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.B-cell genesis in the adult mouse is regulated by a meshwork of stromal-cell components in the bone marrow (1, 2). Establishment of the stromal-cell lines that can support B-cell genesis from multipotent hematopoietic stem cells facilitated identification of the molecules that are required for this process (3-6). Among a number of molecules that are expressed in the stromal-cell lines, interleukin 7 (IL-7) is the first molecule that has been shown to be able to induce proliferation of B-cell precursors (7,8). Initial use of recombinant IL-7 demonstrated that IL-7 can induce proliferation of pre-B cells (8)(9)(10). Subsequent studies further demonstrated that IL-7, when used in combination with the stromal cell lines or the ligand for c-kit, can act on earlier stages of B precursors (11)(12)(13)(14)(15). Besides the proliferative activity on B-precursor cells, it was reported that IL-7 induces proliferation of both mature and immature mouse T cells (16)(17)(18)(19)(20)(21)(22). In fact, studies of mice that received continuous IL-7 injections or bore the IL-7 transgene indicate that elevation of IL-7 results in enormous expansion of both T and B cells (23)(24)(25)(26). Despite all such positive evidence suggesting the role of IL-7 in lymphopoiesis, whether IL-7 is a functional requirement for in vivo lymphocyte production is yet to be determined. To address this question, we have produced an antagonistic monoclonal antibody (mAb) to the high-affinity IL-7 receptor (IL-7Ra) (27) and have investigated its expression and function. Our study demonstrates that IL-7Ra is The publication costs of this article were defrayed in part b...

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-Å resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that VATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V 1 (soluble) and the Vo (membrane bound) domains. Subunit C, attached to the Vo domain, seems to have a socket like function in attaching the central-stalk subunits of the V 1 domain. This architecture seems essential for the reversible association͞dissociation of the V 1 and the Vo domains, unique for V-ATPase activity regulation.T he vacuole-type ATPases (V-ATPases) are commonly found in many organisms involved in a variety of physiological processes (1). V-ATPases in eukaryotic cells (eukaryotic VATPases) translocate protons across the membrane consuming ATP. They reside within intracellular compartments, including endosomes, lysosomes, and secretory vesicles, and within plasma membranes of certain cells including renal intercalated cells, osteoclasts, and macrophages. Eukaryotic V-ATPases are responsible for various cell functions including the acidification of intracellular compartments, renal acidification, born resorption, and tumor metastasis (2).V-ATPase and the F-type ATP synthase (F-ATPase) are evolutionary related and share the rotary mechanism coupling ATP synthesis͞hydrolysis and proton translocation across the membrane (2-4). However, these two types of ATPase show significant differences. Reversible association͞dissociation of the V 1 domain (soluble) and the V o domain (membrane bound) is a unique activity regulation mechanism compared to FATPase (Fig. 1). For example, glucose deprivation has been shown to cause a rapid dissociation of the yeast V-ATPase into free V 1 and V o domains, which is reversible and independent of de novo protein synthesis (5, 6). Similar observations have been reported for Manduca sexta and mammalian complexes (7-9). Subunit composition and structure in the stalk region of VATPase, which connects the V o and V 1 domains, are suggested to be significantly different from those in F-ATPase (10) (Fig. 1). Thus, this region is possibly responsible for the association͞ dissociation of the complex.V-ATPases are also found in archaea and some eubacteria (prokaryotic V-ATPases) (11). The V-ATPase from Thermus thermophilus is solely responsible for aerobic ATP synthesis in this bacteria, which lacks F-ATPase (12). The Thermus VATPase is composed of nine different subunits, which are arranged within the atp operon in the order of G-I-L-E-C-F-A-B-D, which encodes proteins with molecular sizes of 13, 71,8,20,35,12, 64, 54, and 25 kDa, respectively (10) (Fig. 1). This A...

Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow `trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep `figure‐of‐eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2′‐O‐ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 Å resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44‐residue polypeptide chain through a 41‐residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2′‐O‐ribose methyltransferase family are located in the knotting loop and in the knotted carboxy‐terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino‐terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA.

Emerging X-ray free-electron lasers with femtosecond pulse duration enable single-shot snapshot imaging almost free from sample damage by outrunning major radiation damage processes. In bioimaging, it is essential to keep the sample close to its natural state. Conventional high-resolution imaging, however, suffers from severe radiation damage that hinders live cell imaging. Here we present a method for capturing snapshots of live cells kept in a micro-liquid enclosure array by X-ray laser diffraction. We place living Microbacterium lacticum cells in an enclosure array and successively expose each enclosure to a single X-ray laser pulse from the SPring-8 Angstrom Compact Free-Electron Laser. The enclosure itself works as a guard slit and allows us to record a coherent diffraction pattern from a weakly-scattering submicrometre-sized cell with a clear fringe extending up to a 28-nm full-period resolution. The reconstructed image reveals living whole-cell structures without any staining, which helps advance understanding of intracellular phenomena.

Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-A resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water-filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, using polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.