Identification of amino acid residues participating in the energy coupling and proton transport of Streptomyces coelicolor A3(2) H+-pyrophosphatase

AbstractThe H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14–17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr409, Val411, and Gly414 showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe388, Thr389, and Val396 in cytoplasmic loop i were essential for efficient H+ translocation. Ala436 and Pro560 in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H+-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme

Ultrafast spatiotemporal photocarrier dynamics near GaN surfaces studied by terahertz emission spectroscopy

Gallium nitride (GaN) is a promising wide-bandgap semiconductor, and new characterization tools are needed to study its local crystallinity, carrier dynamics, and doping effects. Terahertz (THz) emission spectroscopy (TES) is an emerging experimental technique that can probe the ultrafast carrier dynamics in optically excited semiconductors. In this work, the carrier dynamics and THz emission mechanisms of GaN were examined in unintentionally doped n-type, Si-doped n-type, and Mg-doped p-type GaN films. The photocarriers excited near the surface travel from the excited-area in an ultrafast manner and generate THz radiation in accordance with the time derivative of the surge drift current. The polarity of the THz amplitude can be used to determine the majority carrier type in GaN films through a non-contact and non-destructive method. Unique THz emission excited by photon energies less than the bandgap was also observed in the p-type GaN film

Feasibility of cryopreserved tracheal xenotransplants with the use of short-course immunosuppression

AbstractObjective: We evaluated the feasibility of discordant xenotransplantation of the cryopreserved trachea with intermittent immunosuppression to help solve the shortage of donor tracheas. Methods: Two experiments were performed with heterotopic transplantation models in 14 guinea pigs and 85 rats. So that the minimal dose of FK506 for viable fresh xenografts could be determined, FK506 was given in escalating doses (0, 1.5, 2.5, and 3.5 mg/kg) for recipient animals after xenogeneic transplantation. With the goal of obtaining a long-term survival of the xenografts, the effect of cryopreservation on xenografts was assessed and thereafter different cycles of immunosuppression every third week were evaluated in fresh or cryopreserved xenografts in the second experiment. Results: An FK506 dosage of more than 2.5 mg/kg per day was much more effective than smaller dosages, as demonstrated by morphologic assessment. A higher dosage of FK506 potentially delayed the rejection of xenografts and can thus maintain tracheal xenograft viability for less than 4 weeks in rat recipients. In experiment 2, the cryopreserved xenografts showed less histologic viability than fresh xenografts but greater patency of the lumen. The patency of cryopreserved xenografts was favorably maintained for a longer period than that of fresh xenografts with either the same number or more cycles of immunosuppression. Conclusions: We conclude that the synergistic effect of cryopreservation and adequate intermittent immunosuppression may enable tracheal xenografts to remain viable over longer periods. (J Thorac Cardiovasc Surg 2001;121:241-8

Adaptive data migration scheme with facilitator database and multi-tier distributed storage in LHD

Recent “data explosion” induces the demand for high flexibility of storage extension and data migration. The data amount of LHD plasma diagnostics has grown 4.6 times bigger than that of three years before. Frequent migration or replication between plenty of distributed storage becomes mandatory, and thus increases the human operational costs. To reduce them computationally, a new adaptive migration scheme has been developed on LHD’s multi-tier distributed storage. So-called the HSM (Hierarchical Storage Management) software usually adopts a low-level cache mechanism or simple watermarks for triggering the data stage-in and out between two storage devices. However, the new scheme can deal with a number of distributed storage by the facilitator database that manages the whole data locations with their access histories and retrieval priorities. Not only the inter-tier migration but also the intra-tier replication and moving are even manageable so that it can be a big help in extending or replacing storage equipment. The access history of each data object is also utilized to optimize the volume size of fast and costly RAID, in addition to a normal cache effect for frequently retrieved data. The new scheme has been verified its effectiveness so that LHD multi-tier distributed storage and other next-generation experiments can obtain such the flexible expandability

Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential

Management of Acute Superior Mesenteric Artery Occlusion by Thrombolytic Therapy

Acute occlusion of the superior mesenteric artery (SMA) causes extensive bowel necrosis, resulting in a poor prognosis with an extremely high mortality rate. An 82-year-old woman was admitted to our hospital with the complaint of abdominal pain. She was diagnosed as having acute SMA occlusion by enhanced CT. Five hours from onset, the first thrombolytic therapy with urokinase was performed, but failed to complete thrombolysis and recanalization of peripheral blood flow. An exploratory laparotomy following the first thrombolytic therapy showed a mild ischemic change in the affected intestine and mesentery, but no sign of necrosis. After the laparotomy, local thrombolytic therapy with angiographic evaluation of blood flow at 24, 36 and 48 h from the first thrombolysis was performed. As a result, the residual thrombus disappeared and all branches of the SMA became well visualized. The patient was discharged well without a second-look operation or major bowel resection. Sequential intermittent thrombolytic therapy with meticulous angiographic evaluation of blood flow is effective for early-stage acute SMA occlusion

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering

Ionotropic glutamate receptors in GtoPdb v.2021.3

The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [35, 92, 155]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), 3 TM domains (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy- terminal domain (CTD) [99, 68, 107, 155, 82]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2- see below) has recently been solved at 3.6Å resolution [143] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [71]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [35, 66, 31, 77, 42, 114, 24, 65, 155, 112, 113, 162]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [28]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.NMDA receptorsNMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [41, 25]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [48, 99, 71]. However, more complex tri-heteromeric assemblies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca2+ and is blocked, in a voltage-dependent manner, by Mg2+ such that at resting potentials the response is substantially inhibited.AMPA and Kainate receptorsAMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [43, 103, 153, 64]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [87, 119, 118]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [119, 65, 118]). Kainate receptors may also exhibit 'metabotropic' functions [87, 131]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [118, 88]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl- for their activation [11, 120]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca2+; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [139, 63]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [87, 118]. Native AMPA and kainate receptors displaying differential channel conductances, Ca2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [30, 63, 91]. GluA1-4 can exist as two variants generated by alternative splicing (termed ‘flip’ and ‘flop’) that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [103]. Splice variants of GluK1-3 also exist which affects their trafficking [87, 118]