Corticospinal excitability during observation of basketball free-throw movement: Effects of video playback speed and stimulus timing.

Transcranial magnetic stimulation studies have indicated that action observation (AO) modulates corticospinal excitability. Although a few previous studies have shown that the AO of simple motor movements at a slow playback speed facilitates corticospinal excitability more than that at normal playback speed, it is unclear if this effect occurs during the AO of sport-related complex movements. Therefore, we investigated the changes in the motor evoked potential (MEP) amplitudes of the flexor carpi radialis (FCR) and abductor digiti minimi (ADM) muscles during the AO of a basketball free-throw movement at three different playback speeds (100%, 75%, and 50% speeds). Additionally, we evaluated the effects of stimulus timing (holding the ball vs. releasing the ball for shooting) and motor expertise (expert basketball players vs. novices) on the MEP amplitude during the AO. Our results demonstrated that regardless of motor expertise, the MEP amplitude of the FCR muscle was significantly smaller in the 50% speed condition than in the 100% condition. In the ADM muscle, the MEP amplitude was significantly larger when the ball was held after dribbling than when the ball was released. Therefore, it is suggested that corticospinal excitability in specific muscles during the observation of complex whole-body movements is influenced by video playback speed and stimulus timing.

Soluble Expression in Escherichia coli of a Single-Domain Antibody-Tumor Necrosis Factor α Fusion Protein Specific for Epidermal Growth Factor Receptor.

Tumor-targeted antibody-cytokine fusion proteins, called immunocytokines, are expected to be a useful platform for the development of effective antitumor therapeutic agents; however, their design and cost-efficient production remain as challenges. In this study, we constructed an antibody-cytokine fusion protein (Ia1-TNFα) comprising the single-domain antibody Ia1, which targets epidermal growth factor receptor (EGFR) overexpressed in epithelial tumors and a tumor necrosis factor α (TNFα) domain, which has antitumor activity. Ia1-TNFα was produced in a soluble form by using an Escherichia coli expression system, and after affinity purification of the culture supernatant, an yield of ∼2 mg/L of cell culture was obtained. Gel filtration analysis showed that Ia1-TNFα existed predominantly as a trimer, which is consistent with the multimerization state of TNFα. Ia1-TNFα exhibited approximately 7-fold lower TNFα biological activity than that of TNFα itself. Flow cytometric analysis revealed that Ia1-TNFα specifically bound to EGFR-expressing tumor cells and that its binding activity was higher than that of monovalent Ia1, suggesting that the fusion protein bound to the tumor cells multivalently. Altogether, these results show that fusion of TNFα with a single-domain antibody could be a cost-efficient means of producing antitumor therapeutic agents.

Development of UV Light Irradiation Patterning of Bacteriorhodopsin Thin Films for Biomimetic Functional Devices.

We developed a new patterning method for bacteriorhodopsin (bR) thin films using UV light irradiation. The proton pump function of bR thin films can be deactivated with UV light irradiation. Inactivation of the proton pump function of bR is related to structural changes or photo-bleaching of the retinal in bR using UV light exposure, which was confirmed with absorption and Raman spectroscopy measurements. Utilizing inactivation of the proton pump function with UV light irradiation, we prepared a bR photocell with a stripe-patterned bR thin film and measured its photocurrent response. The new patterning method is applicable to complicated patterning and patterning with a higher spatial resolution, which extends the application of bR thin films as sensor devices.

Conformational difference between two subunits in flavin mononucleotide binding protein dimers from Desulfovibrio vulgaris (MF): molecular dynamics simulation.

The structural and dynamical properties of five FMN binding protein (FBP) dimers, WT (wild type), E13K (Glu13 replaced by Lys), E13R (Glu13 replaced by Arg), E13T (Glu13 replaced by Thr) and E13Q (Glu13 replaced by Gln), were investigated using a method of molecular dynamics simulation (MDS). In crystal structures, subunit A (Sub A) and subunit B (Sub B) were almost completely equivalent in all of the five FBP dimers. However, the predicted MDS structures of the two subunits were not equivalent in solution, revealed by the distances and inter-planar angles between isoalloxazine (Iso) and aromatic amino acids (Trp32, Tyr35 and Trp106) as well as the hydrogen bonding pairs between Iso and nearby amino acids. Residue root of mean square fluctuations (RMSF) also displayed considerable differences between Sub A and Sub B and in the five FBP dimers. The dynamics of the whole protein structures were examined with the distance (RNN) between the peptide N atom of the N terminal (Met1) and the peptide N atom of the C terminal (Leu122). Water molecules were rarely accessible to Iso in all FBP dimers which are in contrast with other flavoenzymes.

Generation and Characterization of Rat Monoclonal Antibodies Against Epidermal Growth Factor Receptor.

Overexpression of the epidermal growth factor receptor (EGFR) gene and dysregulation of EGFR signaling are observed in various cancer cells, and EGFR is a validated target for cancer therapy. In the present study, we report on the generation of two rat anti-EGFR antibodies (clones 2C2D3 and 4H7F4) by using the rat lymph node method. Flow cytometric analysis and immunofluorescence showed that both antibodies specifically bound to EGFR on the surface of cancer cells. Competitive analysis demonstrated that the epitope of each antibody had no overlap with that of the therapeutic anti-EGFR antibody cetuximab. These results suggest that 2C2D3 and 4H7F4 are potentially useful in EGFR-targeted cancer therapy.

Development of a bispecific antibody tetramerized through hetero-associating peptides.

The specific assembly of self-associating peptides can be useful in building a functional antibody complex from small antibody fragments. We have focused on the exceedingly specific heterotetrameric assembly of Lin-2 and Lin-7 (L27) domains, which work as protein-protein interaction modules in many scaffold proteins. Here, we describe a novel method for constructing a highly functional antibody based on the hetero-association of L27 domains. In this study, we used a bacterial expression system to produce a bispecific antibody that was heterotetramerized through L27 domains and that targeted both epidermal growth factor receptor (EGFR) and Fcγ receptor III (FcγRIII or CD16). Gel electrophoresis, mass spectrometry and gel filtration analyses revealed that the constructed recombinant antibody was a disulfide-linked heterotetramer. The tetramerized antibody bound to EGFR and CD16 simultaneously, according to results from flow cytometry and surface plasmon resonance spectroscopy, respectively. Furthermore, we demonstrated that the bispecific antibody showed cytotoxic activity against EGFR-expressing tumor cells by using CD16-positive lymphocytes as effectors, and its cytotoxicity was comparable to that of a commercial therapeutic antibody. Taken together, the results show that our method has high potential for the cost-efficient production of highly active therapeutic antibodies.

Laparoscopic totally extraperitoneal inguinal hernia repair: twenty-seven serious complications after 4565 consecutive operations.

OBJECTIVE: To identify and assess the complications of laparoscopic inguinal hernia treatment with totally extraperitoneal mesh placement (TEP). METHODS: We included patients who had undergone the TEP procedure in a consecutive series of 4565 laparoscopic hernia repairs between January 2001 and January 2011. Inclusion criteria were diagnosis with symptomatic inguinal hernia, including recurrence after inguinal hernia repair and previous surgery in the lower abdomen and pelvis. All patients were 18 years of age or above. Patients with incarcerated hernia in emergency were excluded from the study. RESULTS: A total of 4565 hernias were included in the study. In the group, there were 27 severe complications (0.6%): 12 bleedings (0.25%), two bladder lesions (0.04%), five intestinal obstructions (0.11%), four intestinal perforations (0.09%) one injury to the iliac vein (0.02%), one femoral nerve injury (0.02%), two lesions of vas deferens (0.04%) and two deaths (0.02%) (pulmonary embolism, peritonitis). CONCLUSION: The rate of complications with the TEP procedure is low. Laparoscopic hernia repair technique is reproducible and reliable. In our experience, there are contraindications to the TEP procedure. TEP technique must be meticulous to avoid intraoperative complications (bipolar diathermy). Complications can occur even after the surgeon has gained substantial experience.

Herniorrafia inguinal laparoscópica totalmente extraperitoneal: vinte e sete complicações graves após 4565 operações consecutivas / Laparoscopic totally extraperitoneal inguinal hernia repair: twenty-seven serious complications after 4565 consecutive operations

OBJETIVO: identificar e avaliar as complicações do tratamento da hérnia inguinal com a colocação de tela totalmente extraperitoneal. MÉTODOS: Foram incluídos, em uma série consecutiva de 4565 reparos de hérnia laparoscópica, pacientes que haviam sido submetidos ao procedimento TEP entre janeiro de 2001 e janeiro de 2011. Os critérios de inclusão foram: diagnóstico com hérnia inguinal sintomática, incluindo recorrência após correção de hérnia inguinal e cirurgia prévia em abdômen inferior e pelve. Todos os pacientes > 18 anos de idade. Pacientes com hérnia encarcerada na urgência foram excluídos do estudo. RESULTADOS: Um total de 4565 hérnias foram incluídas no estudo. Ocorreram 27 complicações graves (0,6%): 12 hemorragias (0,25%), duas lesões da bexiga (0,04%), cinco oclusões (0,11%), quatro perfurações intestinais (0,09%), uma lesão da veia ilíaca (0,02%), uma lesão do nervo femoral (0,02%), duas lesões dos vasos deferentes (0,04%) e dois óbitos (0,02%) (embolia pulmonar, peritonite). CONCLUSÃO: A taxa de complicações com o procedimento TEP é baixa. Correção de hérnia laparoscópica é uma técnica reprodutível e confiável. Em nossa experiência, existem contraindicações para o procedimento de TEP. A técnica TEP deve ser minuciosa para evitar complicações intraoperatórias (diatermia bipolar). As complicações podem ocorrer mesmo após o cirurgião ter adquirido experiência substancial.

OBJECTIVE: To identify and assess the complications of laparoscopic inguinal hernia treatment with totally extraperitoneal mesh placement (TEP). METHODS: We included patients who had undergone the TEP procedure in a consecutive series of 4565 laparoscopic hernia repairs between January 2001 and January 2011. Inclusion criteria were diagnosis with symptomatic inguinal hernia, including recurrence after inguinal hernia repair and previous surgery in the lower abdomen and pelvis. All patients were 18 years of age or above. Patients with incarcerated hernia in emergency were excluded from the study. RESULTS: A total of 4565 hernias were included in the study. In the group, there were 27 severe complications (0.6%): 12 bleedings (0.25%), two bladder lesions (0.04%), five intestinal obstructions (0.11%), four intestinal perforations (0.09%) one injury to the iliac vein (0.02%), one femoral nerve injury (0.02%), two lesions of vas deferens (0.04%) and two deaths (0.02%) (pulmonary embolism, peritonitis). CONCLUSION: The rate of complications with the TEP procedure is low. Laparoscopic hernia repair technique is reproducible and reliable. In our experience, there are contraindications to the TEP procedure. TEP technique must be meticulous to avoid intraoperative complications (bipolar diathermy). Complications can occur even after the surgeon has gained substantial experience.

Crystal structure analysis of the translation factor RF3 (release factor 3).

The bacterial translational GTPases release factor RF3 promotes translation termination by recycling RF1 or RF2. Here, we present the crystal structures of RF3 complexed with GDP and guanosine 3',5'-(bis)diphosphate (ppGpp) at resolutions of 1.8 and 3.0Å, respectively. ppGpp is involved in the so-called "stringent response" of bacteria. ppGpp binds at the same site as GDP, suggesting that GDP and ppGpp are two alternative physiologically relevant ligands of RF3. We also found that ppGpp decelerates the recycling of RF1 by RF3. These lines of evidence suggest that RF3 functions both as a cellular metabolic sensor and as a regulator.

[A long-surviving patient with unresectable advanced gastric cancer treated with S-1 and biweekly paclitaxel combination chemotherapy as second-line treatment].

A 59-year-old man with type 3 gastric cancer(signet-ring cell carcinoma)underwent simple laparotomy because of peritoneal dissemination.S -1/CDDP was started.Since the icterus of Grade 2 had appeared after 2 courses, S-1 and biweekly paclitaxel combination chemotherapy was started as second-line treatment.Throughout treatment, there was no adverse event, and this regimen was continued for 14 courses(25 months).He died 32 months after his first visit.S -1/PTX may play an important role as second-line chemotherapy for patients with unresectable advanced gastric carcinoma.

Effects of the disappearance of one charge on ultrafast fluorescence dynamics of the FMN binding protein.

Crystal structures of E13T (Glu13 was replaced by Thr13) and E13Q (Glu13 was replaced by Gln13) FMN binding proteins (FMN-bp) from Desulfovibrio vulgaris, strain Miyazaki F, were determined by the X-ray diffraction method. Geometrical factors related to photoinduced electron transfer from Trp32, Tyr35, and Trp106 to the excited isoalloxazine (Iso*) were compared among the three forms of FMN-bp. The rate of ET is considered to be fastest from Trp32 to Iso* in FMN-bp and then from Tyr35 and Trp106. The distances between Iso and Trp32 did not change appreciably (0.705-0.712 nm) among WT, E13T, and E13Q FMN-bps, though the distances between Iso and Tyr35 or Trp106 became a little shorter by ca. 0.01 nm in both mutated FMN-bps. The distances between the residue at 13 and the ET donors or acceptor in the mutated proteins, however, changed markedly, compared to WT. Hydrogen bonding pairs and distances between Iso and surrounding amino acids were not modified when Glu13 was replaced by Thr13 or Gln13. Effects of elimination of ionic charge at Glu13 on the ultrafast fluorescence dynamics in E13T and E13Q were investigated comparing to WT, by means of a fluorescence up-conversion method. Fluorescence lifetimes were tau(1) = 107 fs (alpha(1) = 0.86), tau(2) = 475 fs (alpha(2) = 0.12), and tau(3) = 30 ps (alpha(3) = 0.02) in E13T and tau(1) = 134 fs (alpha(1) = 0.85), alpha(2) = 746 fs (alpha(2) = 0.12), and tau(3) = 30 ps (alpha(3) = 0.03) in E13Q, which are compared to the reported lifetimes in WT, tau(1) = 168 fs (alpha(1) = 0.95) and alpha(2) = 1.4 ps (alpha(2) = 0.05). Average lifetimes (tau(AV) = Sigma(i=1)(2or3)alpha(i)tau(i)) were 0.75 ps in E13T, 1.10 ps in E13Q, and 0.23 ps in WT, which implies that tau(AV) was 3.3 times longer in E13T and 4.8 times longer in E13Q, compared to WT. The ultrafast fluorescence dynamics of WT did not change when solvent changed from H(2)O to D(2)O. Static ET rates (inverse of average lifetimes) were analyzed with static structures of the three systems of FMN-bp. Net electrostatic (ES) energies of Iso and Trp32, on which ET rates depend, were 0.0263 eV in WT, 0.322 eV in E13T, and 0.412 eV in E13Q. The calculated ET rates were in excellent agreement with the observed ones in all systems.

Schwannoma of the gallbladder: report of a case.

We report a schwannoma of the gallbladder in a 58-year-old man who was diagnosed as cholecystolithiasis. He presented with recurrent episodes of abdominal pain in the right upper quadrant. The abdominal computed tomography scan and ultrasonography revealed stones about 15 mm in diameter in the gallbladder. Under the diagnosis of cholecystolithiasis, laparoscopic cholecystectomy was performed. The resected specimen showed chronic cholecystitis and no suspicion of neoplasm. Pathological examination revealed that the tumor consist of spindle cells without atypical appearance at small area of fundus. Immunohistologically, tumor cells were positive for S-100 protein and negative for alpha-SMA and c-kit, the lesion was diagnosed as schwannoma.

Structure analysis of the flavoredoxin from Desulfovibrio vulgaris Miyazaki F reveals key residues that discriminate the functions and properties of the flavin reductase family.

The crystal structure of flavoredoxin from Desulfovibrio vulgaris Miyazaki F was determined at 1.05 A resolution and its ferric reductase activity was examined. The aim was to elucidate whether flavoredoxin has structural similarity to ferric reductase and ferric reductase activity, based on the sequence similarity to ferric reductase from Archaeoglobus fulgidus. As expected, flavoredoxin shared a common overall structure with A. fulgidus ferric reductase and displayed weak ferric reductase and flavin reductase activities; however, flavoredoxin contains two FMN molecules per dimer, unlike A. fulgidus ferric reductase, which has only one FMN molecule per dimer. Compared with A. fulgidus ferric reductase, flavoredoxin forms three additional hydrogen bonds and has a significantly smaller solvent-accessible surface area. These observations explain the higher affinity of flavoredoxin for FMN. Unexpectedly, an electron-density map indicated the presence of a Mes molecule on the re-side of the isoalloxazine ring of FMN, and that two zinc ions are bound to the two cysteine residues, Cys39 and Cys40, adjacent to FMN. These two cysteine residues are close to one of the putative ferric ion binding sites of ferric reductase. Based on their structural similarities, we conclude that the corresponding site of ferric reductase is the most plausible site for ferric ion binding. Comparing the structures with related flavin proteins revealed key structural features regarding the discrimination of function (ferric ion or flavin reduction) and a unique electron transport system.

Cloning and expression of the MutM gene from obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).

The gene encoding a MutM from Desulfovibrio vulgaris (Miyazaki F) was cloned and expressed in Escherichia coli. A 5.9-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by XhoI and PvuII, contained a MutM gene and other open reading frames. The nucleotide sequence of the MutM gene indicated that the protein was composed of 336 amino acids. The amino-acid sequence deduced from the MutM gene was highly homologous with the MutM of other bacteria; however an additional insert consisted of 64 amino acids. An expression system for the MutM gene under the control of the T7 promoter was constructed in E. coli. From the kinetic analysis results, the purified His-tagged MutM showed 8-oxoguanine-DNA glycosylase activity comparable with that of MutM from E. coli. In this study, the amounts of mRNA and protein for MutM were scant in the D. vulgaris (Miyazaki F). MutM activity may be induced by oxidative stress. However, its induction may not be frequently generated because sulfate-reducing bacteria generally grow in anaerobic conditions. MutM might play a role in the protection against the mutagenicity of oxygen when oxygen stress exceeded the capacity of the defense systems against oxygen toxicity.

Structure-activity relationship of an antisense oligonucleotide-two Cu(II) complex conjugate as an artificial ribonuclease.

We previously demonstrated that an antisense 2'-O-methyloligonucleotide, with two terpyridine*Cu(II) complexes at contiguous internal sites, was highly active as a sequence-specific artificial ribonuclease. Two kinds of terpyridine-linked uridine derivatives (Ut and tU(L)) were used for its construction, and the residue on the 5'-side (Ut) was the derivative with terpyridine attached to the 2'-oxygen via a short linker arm. To examine the structure-activity relationship of this type of RNA cleaver, we synthesized an analogous RNA cleaver with the inosine counterpart (It) on the 5'-side, since inosine can base-pair with A, U or C. Using the RNA cleavers and the RNA oligomer substrates, we examined the effect of base-pair formation at the Ut (or It) and tU(L) sites on the activities of the cleavers. The cleavage reactions revealed that, for this type of RNA cleaver, a base-pair at the 5'-side and no base-pair at the 3'-side were required for high activity. In addition, for the 5'-side-It residue, a normal base-pair (I-C pair) was needed.

Crystallization and preliminary X-ray crystallographic study of flavoredoxin from Desulfovibrio vulgaris Miyazaki F.

Flavoredoxin from Desulfovibrio vulgaris Miyazaki F has been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with 10%(w/v) PEG 8000, 0.2 M zinc acetate and 100 mM MES pH 6.0. The diffraction pattern of the crystal extended to 1.05 A resolution under cryogenic conditions. The space group was determined to be P3(1)21, with unit-cell parameters a = b = 53.35, c = 116.22 A. Phase determination was carried out by the SAD method using methylmercuric chloride.

Crystallization and preliminary X-ray analysis of a class II release factor RF3 from a sulfate-reducing bacterium.

Class II release factor 3 (RF3) from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F, which promotes rapid dissociation of a class I release factor, has been overexpressed, purified and crystallized in complex with GDP at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to 1.8 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belongs to space group P1, with unit-cell parameters a = 47.39, b = 82.80, c = 148.29 A, alpha = 104.21, beta = 89.78, gamma = 89.63 degrees . The asymmetric unit contains four molecules of the RF3-GDP complex. The Matthews coefficient was calculated to be 2.3 A(3) Da(-1) and the solvent content was estimated to be 46.6%.

Development of site-specific artificial ribonucleases.

Recently we found that an antisense 2'-O-methyloligonucleotide, with two terpyridine*Cu(II) complexes at contiguous internal sites, was highly active as a site-specific (sequence-specific) artificial ribonuclease, with the activity derived from the cooperative action of the complexes. Two kinds of terpyridine-linked nucleosides were used for the construction of the RNA cleaver, including a uridine derivative with terpyridine attached to the 2'-oxygen via a short linker arm. In order to explore more efficient cleavers (practical cleavers), we have constructed a structurally similar cleaver (18-mer), but containing a novel 2'-carbon-branched uridine with a terpyridine group instead of the aforementioned 2'-oxygen-modified uridine. The reaction of a 10-fold excess of the target RNA 24-mer with the new agent, in the presence of Cu(II) ions, and at pH 7.5 and 37 degrees C, revealed that the substrate was cleaved in 92% yield after 5 h. Under similar conditions, the previous cleaver was less active and the cleavage yield was 61%.

Comparison between ultrafast fluorescence dynamics of FMN binding protein from Desulfovibrio vulgaris, strain Miyazaki, in solution vs crystal phases.

Ultrafast fluorescence dynamics of FMN binding protein (FBP) from Desulfobivrio vulgaris, strain Miyaxaki F, were compared in solution and crystal phases. Fluorescence lifetimes of FBP were 167 fs (96%) and 1.5 ps (4%) in solution (tau(av) = 220 fs), and 730 fs (60%) and longer than 10 ps (40%) in crystals (tau(av) = 4.44 ps). The quenching of the fluorescence of flavin in the protein was considered to be due to photoinduced electron transfer (ET) from Trp or Tyr to the excited isoalloxazine (Iso) nearby. The average lifetime was 20 times longer in crystal vs in solution. Averaged distances between Iso and nearby Trp-32, Tyr-35, and Trp-106 were 8.42, 7.36, and 8.15 A in solution, respectively (obtained by NMR spectroscopy), and 7.05, 7.72, and 8.49 A in crystal, respectively (obtained by X-ray crystallography). The prolonged lifetime in crystal cannot be elucidated by the change in the distances between the states. It was suggested that the longer lifetime in crystal was ascribed to the absence of water molecules around FBP with rapid motional freedom, which may be the driving force for the ET in flavoproteins.

Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.

A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein. The apo-form of the protein was synthesized efficiently in the absence of FMN. The purified apo-form could be correctly converted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism-spectra studies suggested that the FMN-binding site of the apo-form is open as compared with the holo-form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3-azido-L-tyrosine at the Tyr35 residue in the FMN-binding site. The authors optimized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triarylphosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 residue in the apo-form is open to solvent.