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Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. Here we report the interaction between the HBx BH3-like motif and Bcl-xL, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-xL, with a binding affinity of 89 μM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-xL binding using three mutants. The W120A mutant showed weaker binding affinity (294 μM) to Bcl-xL, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-xL binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.

Despite accumulating evidence that protein dynamics is indispensable for understanding the structural basis of biological activities, it remains challenging to visualize the spatial description of the dynamics and to associate transient conformations with their molecular functions. We have developed a new NMR protein structure determination method for the inference of multi-state conformations using multiple types of NMR data, including paramagnetic NMR and residual dipolar couplings, as well as conventional NOEs. Integration of these data in the structure calculation permits delineating accurate ensemble structures of biomacromolecules. Applying the method to the protein yeast ubiquitin hydrolase 1 (YUH1), we find large dynamics of its N-terminus and crossover loop surrounding the active site for ubiquitin-recognition and proteolysis. The N-terminus gets into and out of the crossover loop, suggesting their underlying functional significance. Our results, including those from biochemical analysis, show that large motion surrounding the active site contributes strongly to the efficiency of the enzymatic activity.

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 μM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-x

Several preconditioners are proposed for improving the convergence rate of the iterative method derived from splitting. In this paper, the comparison theorem of preconditioned iterative method for regular splitting is proved. And the convergence and comparison theorem for any preconditioner are indicated. This comparison theorem indicates the possibility of finding new preconditioner and splitting. The purpose of this paper is to show that the preconditioned iterative method yields a new splitting satisfying the regular or weak regular splitting. And new combination preconditioners are proposed. In order to denote the validity of the comparison theorem, some numerical examples are shown.

An image-intensified, camera-based track imaging system has been developed to measure the tracks of ions in a scintillator block. To study the performance of the detector unit in the system, two types of scintillators, a dosimetrically tissue-equivalent plastic scintillator EJ-240 and a CsI(Tl) scintillator, were separately irradiated with carbon ion ( 12 C) beams of therapeutic energy from HIMAC at NIRS. The images of individual ion tracks in the scintillators were acquired by the newly developed track imaging system. The ranges reconstructed from the images are reported here. The range resolution of the measurements is 1.8 mm for 290 MeV/u carbon ions, which is considered a significant improvement on the energy resolution of the conventional Δ E / E method. The detector is compact and easy to handle, and it can fit inside treatment rooms for in-situ studies, as well as satisfy clinical quality assurance purposes.

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with “off-target” molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

Non-detergent sulfobetaines (NDSBs) are a new group of small, synthetic protein stabilizers, which have advantages over classical compatible osmolytes, such as polyol, amines, and amino acids: they do not increase solution viscosity, unlike polyols, and they are zwitterionic at all pH ranges, unlike amines and amino acids. NDSBs also facilitate the crystallization and refolding of proteins. The mechanism whereby NDSBs exhibit such activities, however, remains elusive. To gain insight into this mechanism, we studied, using nuclear magnetic resonance (NMR), the effects of dimethylethylammonium propane sulfonate (NDSB-195) on the dynamics of ubiquitin, on which a wealth of information has been accumulated. By analyzing the line width of amide proton resonances and the transverse relaxation rates of nitrogen atoms, we found that NDSB-195 enhances the microsecond-millisecond dynamics of a β4 -α2 loop of ubiquitin. Although those compounds that enhance protein dynamics are generally considered to destabilize protein molecules, NDSB-195 enhanced the stability of ubiquitin against guanidium chloride denaturation. Thus, the simultaneous enhancement of stability and flexibility by a single compound can be attained.

Monte Carlo simulation is the most accurate tool for calculating dose distributions. In particular, the Electron Gamma shower computer code has been widely used for multi-purpose research in radiotherapy, but Monte Carlo GEANT4 (GEometry ANd Tracking) is rare for radiotherapy with photon beams and needs to be verified further under various irradiation conditions, particularly multi-leaf collimator-based intensity-modulated radiation therapy (MLC-based IMRT). In this study, GEANT4 was used for modeling of a 6 MV linac for dynamic MLC-based IMRT. To verify the modeling of our linac, we compared the calculated data with the measured depth-dose for a 10 × 10 cm(2) field and the measured dose profile for a 35 × 35 cm(2) field. Moreover, 120 MLCs were modeled on the GEANT4. Five tests of MLC modeling were performed: (I) MLC transmission, (II) MLC transmission profile including intra- and inter-leaf leakage, (III) tongue-and-groove leakage, (IV) a simple field with different field sizes by use of MLC and (V) a dynamic MLC-based IMRT field. For all tests, the calculations were compared with measurements of an ionization chamber and radiographic film. The calculations agreed with the measurements: MLC transmissions by calculations and measurements were 1.76 ± 0.01 and 1.87 ± 0.01 %, respectively. In gamma evaluation method (3 %/3 mm), the pass rates of the (IV) and (V) tests were 98.5 and 97.0 %, respectively. Furthermore, tongue-and-groove leakage could be calculated by GEANT4, and it agreed with the film measurements. The procedure of commissioning of dynamic MLC-based IMRT for GEANT4 is proposed in this study.

M-ficolin, which forms trimer-based multimers, is a pathogen-recognition protein in the innate immune system, and it binds to ligands through its fibrinogen-like (FBG) domain. As the first step toward the elucidation of the molecular basis for pathogen-recognition by the M-ficolin multimers, we assigned the backbone resonances of the monomeric mutant of the M-ficolin FBG domain, recombinantly expressed by Brevibacillus choshinensis. Like the wild-type trimeric FBG domain, the monomeric FBG domain also requires His251, His284 and His297 for the ligand-binding activity, as judged by mutational analyses using zonal affinity chromatography. The secondary structure predicted by the backbone resonance assignments is similar to that of the trimeric FBG domain in the crystal, indicating that the monomeric FBG domain is folded correctly to perform its function.

The purpose of paper is to analyze the behavior of the error in the iterative method. Especially, we are interested in the classical iterative method such as SOR method and its preconditioning techniques to solve the linear system \(A{u}={q}\). In order to accelerate convergence, many researchers proposed several preconditioners [4, 5, 6, 7, 8]. There is also preconditioner available for both classical iterative and Krylov subspace methods. We focus on the behavior of error to find a good preconditioner. We treat difference equation derived from partial differential equation(PDE), because the coefficient matrix given by using difference approximation is easy to investigate. By examining the behavior of the error, we choose an effective preconditioner, and show the numerical results.

Kurtoxin is a 63-amino acid polypeptide isolated from the venom of the South African scorpion Parabuthus transvaalicus. It is the first and only peptide ligand known to interact with Cav3 (T-type) voltage-gated Ca(2+) channels with high affinity and to modify the voltage-dependent gating of these channels. Here we describe the nuclear magnetic resonance (NMR) solution structure of kurtoxin determined using two- and three-dimensional NMR spectroscopy with dynamical simulated annealing calculations. The molecular structure of the toxin was highly similar to those of scorpion α-toxins and contained an α-helix, three β-strands, and several turns stabilized by four disulfide bonds. This so-called "cysteine-stabilized α-helix and β-sheet (CSαβ)" motif is found in a number of functionally varied small proteins. A detailed comparison of the backbone structure of kurtoxin with those of the scorpion α-toxins revealed that three regions [first long loop (Asp(8)-Ile(15)), β-hairpin loop (Gly(39)-Leu(42)), and C-terminal segment (Arg(57)-Ala(63))] in kurtoxin significantly differ from the corresponding regions in scorpion α-toxins, suggesting that these regions may be important for interacting with Cav3 (T-type) Ca(2+) channels. In addition, the surface profile of kurtoxin shows a larger and more focused electropositive patch along with a larger hydrophobic surface compared to those seen on scorpion α-toxins. These distinct surface properties of kurtoxin could explain its binding to Cav3 (T-type) voltage-gated Ca(2+) channels.

Purpose: Microdosimetry has been developed for the evaluation of radiation quality, and single-event dose-mean lineal energy y − D is well-used to represent the radiation quality. In this study, the changes of the relative biological effectiveness (RBE) values under the therapeutic conditions using a 6 MV linac were investigated with a microdosimetric method. Methods: The y − D values under the various irradiation conditions for x-rays from a 6 MV linac were measured with a tissue-equivalent proportional counter (TEPC) at an extremely low dose rate of a few tens of μGy/min by decreasing the gun grid voltage of the linac. According to the microdosimetric kinetic model (MK model), the RBEMK values for cell killing of the human salivary gland (HSG) tumor cells can be derived if the y − D values are obtained from TEPC measurements. The Monte Carlo code GEANT4 was also used to calculate the photon energy distributions and to investigate the changes of the y − D values under the various conditions. Results: The changes of the y − D values were less than approximately 10% when the field size and the depth in a phantom varied. However, in the measurements perpendicular to a central beam axis, large changes were observed between the y − D values inside the field and those outside the field. The maximum increase of approximately 50% in the y − D value outside the field was obtained compared with those inside the field. The GEANT4 calculations showed that there existed a large relative number of low energy photons outside of the field as compared with inside of the field. The percentages of the photon fluences below 200 keV outside the field were approximately 40% against approximately 8% inside the field. By using the MK model, the field size and the depth dependence of the RBEMK values were less than approximately 2% inside the field. However, the RBEMK values outside the field were 6.6% higher than those inside the field. Conclusions: The increase of the RBEMK values by 6.6% outside the field was observed. This increase is caused by the change of the photon energy distributions, especially the increase of the relative number of low energy photons outside the field.

An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-d-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.

Protein translocation across the bacterial cell membrane is mediated by the SecYEG translocon and is enhanced by a membrane protein called SecDF, the function of which was unknown. In this study, Osamu Nureki and colleagues present a structural and functional analysis of SecDF. They show that it has 12 transmembrane domains and two major periplasmic domains (P1 and P4), and propose that SecDF functions as a membrane-integrated chaperone, powered by the proton motive force to perform protein translocation. Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase1,2,3,4, is enhanced by proton motive force5,6 and membrane-integrated SecDF7,8,9, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis4,10,11,12,13. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 A resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.

The purpose of this article is to analyze several Lie algebras associated to “orbit configuration spaces” obtained from a group G acting freely, and properly discontinuously on the upper half-plane H 2 . The Lie algebra obtained from the descending central series for the associated fundamental group is shown to be isomorphic, up to a regrading, to 1. the Lie algebra obtained from the higher homotopy groups of analogous constructions associated to H 2 × C q modulo torsion, as well as 2. the Lie algebra obtained from horizontal chord diagrams for surfaces. The resulting Lie algebras are similar to those studied in [T. Kohno, Linear representations of braid groups and classical Yang-Baxter equations, Contemp. Math. 78 (1988) 339–363; T. Kohno, Vassiliev invariants and de Rham complex on the space of knots, Contemp. Math. 179 (1994) 123–138; T. Kohno, Elliptic KZ system, braid groups of the torus and Vassiliev invariants, Topology and its Applications 78 (1997) 79–94; D.C. Cohen, Monodromy of fiber-type arrangements and orbit configuration spaces, Forum Math. 13 (2001) 505–530; F.R. Cohen, M. Xicotencatl, On orbit configuration spaces associated to the Gaussian integers: homology and homotopy groups, Topology Appl. 118 (2002) 17–29; E. Fadell and S. Husseini, The space of loops on configuration spaces and the Majer-Terracini index, Topol. Methods Nonlinear Anal. J. Julius Schauder Center 11 (1998), 249–271; E. Fadell and S. Husseini, Geometry and Topology of Configuration Spaces, in: Springer Monographs in Mathematics, Springer-Verlag, 2001; F.R. Cohen and T. Sato, On groups of morphisms of coalgebras, (submitted for publication)]. The structure of a related graded Poisson algebra defined below and obtained from an analogue of the infinitesimal braid relations parametrized by G is also addressed.

Although detergents have been widely used in G-protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G-proteins. Hence, we analysed the effects of detergents on guanine nucleotide exchange reactions of Galpha(i1). Lubrol PX, a non-ionic detergent, which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPgammaS binding from/to Galpha in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G-proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPgammaS binding, and the binding rate constant was decreased by three orders of magnitude in the presence of 2% cholate. These results demonstrate that the guanine nucleotide exchange reactions of Galpha(i1) are drastically modulated by detergents differently depending on the type and the state (monomeric or micellar) of the detergents and that dissociation of GDP from Galpha(i1) does not necessarily lead to immediate binding of GTP to Galpha(i1) in some cases. These effects of detergents on G-proteins must be taken into account in G-protein experiments.

The aim of this study is to investigate p16INK4a expression by immunocytochemistry for ascites in advanced ovarian cancer and explore the possibility to predict chemotherapeutic response and prognosis. The immunocytochemical study was performed on cytology of ascites obtained from 37 Stage III or Stage IV ovarian cancer patients with measurable disease before platinum/taxane-based first-line chemotherapy following primary cytoreductive surgery or as neoadjuvant chemotherapy. Twenty-one of 21 (100%) responders and 6 of 16 (44%) nonresponders showed p16INK4a immunopositivity (p < 0.001). Immunopositivity was frequently observed in serous adenocarciomas (17 of 18, 94%). Overall survival was significantly better in immunopositive cases compared with immunonegative cases (p = 0.0006). For subcellular localization, cytoplasm was diffusely positive in immunopositive cases (n = 27), 12 of which showed stronger nuclear immunostaining and demonstrated superior overall survival. In vitro expression of p16INK4a protein was also examined for both parent chemosensitive and acquired chemoresistant ovarian cancer cell lines. Chemosensitive KF28 parent cells showed stronger nuclear staining compared with chemoresistant KFr13Tx cells showing stronger cytoplasmic staining by immunocytochemistry, which were also confirmed by western blotting. Our data suggest that p16INK4a expression in cytology of ascites is a candidate marker in prediction of the primary response to chemotherapy and prognosis.

Red blood cell protein 4.1 (4.1R, an 80 kDa isoform),1 one of the major erythrocyte cytoskeletal proteins, forms two major ternary complexes in red blood cells, 4.1Ractin-spectrin and 4.1R-p55-glycophorin C (GPC), and plays important roles in maintaining cell morphology and membrane mechanical properties.1–7 This protein consists of four functional domains: the FERM (4.1 and ezrin/radixin/moesin)8 domain (also termed the N-terminal 30 kDa membrane-binding domain), the 16 kDa regulatory domain, the spectrin-actin-binding (SAB) domain, and the C-terminal 22/24 kDa domain. The 4.1R FERM domain is a multifunctional domain that forms a cloverleaf-like fold containing three structural and functional domains (N-lobe, a-lobe, and C-lobe domains).9 In the 4.1R-p55-GPC ternary complex, the 4.1R FERM a-lobe domain interacts with the N-terminal cytoplasmic domain of GPC, the 4.1R FERM C-lobe10 and/or N-lobe domain11 interacts with the p55 HOOK domain, and the p55 PDZ domain interacts with the C-terminal cytoplasmic domain of GPC.10,12–14 We previously examined the specific binary interaction between the p55 PDZ domain and the C-terminal cytoplasmic domain of GPC by NMR, as the first step to obtain structural insights into the 4.1R-p55-GPC ternary complex.15,16 Our NMR studies clearly revealed how the p55 PDZ domain interacts with two key C-terminal residues, Tyr126 and Ile128, of GPC at the atomic level.16 We have now focused on the binary interaction between the 4.1R FERM a-lobe domain and the N-terminal cytoplasmic domain of GPC, as the second step toward our goal. In the binary interaction between 4.1R and GPC, the 4.1R-binding site on GPC reportedly corresponds to the Nterminal 12 amino acid residues of its cytoplasmic domain (residues 82–93), and the RHK (residues Arg86-His87Lys88) motif is probably crucial for binding the 4.1R FERM a-lobe domain.13 In contrast, the GPC-binding site on the 4.1R FERM domain reportedly corresponds to the sequence encoded by exon 8 (residues Tyr94-Arg166) in the 4.1R alobe domain, and the ELEE motif (residues Glu153Glu156) is considered to be a good candidate for the GPC-binding site.9,10 However, there is no experimental evidence that the ELEE motif contributes to the interaction with the N-terminal cytoplasmic domain of GPC. To better understand this interaction mode, we prepared the 4.1R FERM a-lobe domain (residues Asp83-Tyr187) and performed an NMR analysis of this domain with the N-terminal cytoplasmic domain of GPC. Here, we report the first NMR-derived structure of the 4.1R FERM a-lobe domain and propose a new GPC-binding site on the 4.1R FERM a-lobe domain, based on our NMR experiments.

Jumonji/JARID2 (Jmj) is a transcriptional repressor protein, which plays important roles in development, cell growth and gene expression.1–3 For example, recent studies have revealed that Jmj represses the expression of cyclin D1, a key component of the cell cycle machinery, by inducing histone H3 lysine 9 (H3-K9) methylation in the complex with G9a and GLP, which are H3-K9 methyltransferase proteins.4,5 In addition, Jmj has been shown to repress atrial natriuretic factor (ANF) gene expression, by interacting with the transcriptional activities of the cardiac transcription factors, Nkx2.5 and GATA4.6 Therefore, it is believed that Jmj may use different regulatory mechanisms with multiple target genes. The Jmj protein consists of at least six structural and functional domains [Fig. 1(A)]: the nuclear localization signal (NLS) domain, the transcriptional repressor domain, the JmjN domain, the AT-rich interaction domain (ARID), the JmjC domain and the Zinc finger-like C5HC2 domain.3 The ARID domain7,8 is a distinct DNA-binding module and binds to DNA through a long loop that connects the helices of a helix-turn-helix (HTH) motif, and therefore this HTH motif is called as a noncanonical HTH motif.9,10 ARID domains have both functional and structural diversities.7,8 For example, the Dri-ARID9,10 and Mrf2-ARID11 domains interact with their specific AT-rich DNA sequences, while the RBP2-ARID domain binds to a GC-rich CCGCC DNA sequence.12 Furthermore, the SWI1-ARID domain reportedly interacts with nonspecific DNA sequences.13 So far, three structural classes have been represented: (i) the minimal ARID domain (six a-helices), (ii) both the Nand C-terminal extended ARID domain and (iii) the N-terminal extended ARID domain. However, the structure of the Cterminal extended ARID domain has not been determined yet. We previously performed NMR analyses of the Jmj-ARID domain consisting of residues Leu615Lys730.14 The secondary structure prediction from the NMR data revealed that the Jmj-ARID domain may possess an additional helix at the C-terminus. Thus, the JmjARID domain seems to be a good candidate to represent the C-terminal ARID domain. Here we report the first NMR-derived structure of the ARID of Jumonji/JARID2.

Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc (N-acetylglucosamine), on pathogen surfaces. From previous structural and functional studies of the FD1 (M-ficolin fibrinogen-like domain), we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pKa of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M., Kondo, S., Sugio, S. and Kohno, T. (2007) J. Biol. Chem. 282, 3889–3895]. To investigate the origins of the pH dependency, mutational analyses were performed on FD1 expressed by Brevibacillus choshinensis. The GlcNAc binding study of a series of single histidine mutants of FD1 demonstrated that His251, His284 and His297 are required for the activity, and thus we concluded that the three histidines are the origins of the pH dependency of FD1. Monomeric mutants of FD1 show weaker affinity for the ligand than the trimeric wild-type, indicating that trimer formation confers high avidity for the ligand. In addition, analyses of the GlcNAc association and dissociation of FD1 provided evidence that FD1 always exchanges between the active and non-active states with the pH-dependent populations in solution. The biological roles of the histidine-regulated conformational equilibrium of M-ficolin are discussed in terms of the self and non-self discrimination mechanism.

In recent years, a number of preconditioners have been applied to linear systems [A.D. Gunawardena, S.K. Jain, L. Snyder, Modified iterative methods for consistent linear systems, Linear Algebra Appl. 154–156 (1991) 123–143; T. Kohno, H. Kotakemori, H. Niki, M. Usui, Improving modified Gauss–Seidel method for Z-matrices, Linear Algebra Appl. 267 (1997) 113–123; H. Kotakemori, K. Harada, M. Morimoto, H. Niki, A comparison theorem for the iterative method with the preconditioner (I+Smax), J. Comput. Appl. Math. 145 (2002) 373–378; H. Kotakemori, H. Niki, N. Okamoto, Accelerated iteration method for Z-matrices, J. Comput. Appl. Math. 75 (1996) 87–97; M. Usui, H. Niki, T.Kohno, Adaptive Gauss-Seidel method for linear systems, Internat. J. Comput. Math. 51(1994)119–125 [10]]. Since these preconditioners are constructed from the elements of the upper triangular part of the coefficient matrix, the preconditioning effect is not observed on the nth row of matrix A. In the present paper, in order to deal with this drawback, we propose a new preconditioner. In addition, the convergence and comparison theorems of the proposed method are established. Simple numerical examples are also given, and we show that the convergence rate of the proposed method is better than that of the optimum SOR.

Ubiquitin–peptide fusion protein system enables preparation of stable isotope labeled peptides through the expression of the protein in E. coli cells in labeled media (Kohno et al. (1998) J Biomol NMR 12:109–121). Advantages of the system over others include: very specific cleavage of the bond between ubiquitin and target peptide moieties by yeast ubiquitin hydrolase and low cost for the protease which can also be expressed in E. coli cells. The former point is particularly important since other frequently used proteases, such as factor Xa and thrombin, often show non-specific cleavages at sites unexpected from their nominal specificities. We improved the yield of the peptide by adapting the codon usage of ubiquitin gene for the expression in E. coli cells, by using RNase E-deficient host strains, and by modifying purification procedure. The yield of mastoparan-X was increased threefold by these modifications. We also succeeded in the preparation of labeled magainin 2, an antimicrobial peptide that could not be expressed at all by the previous method, by choosing host strains and culture media. The HSQC signals of the 15N-labeled magainin 2 in an aqueous solution were completely resolved in spite of the severe overlap of the 1D proton signals, confirming that the stable isotope labeling is quite useful for analysis of peptides.

The molecular dissection of human MCM2, a constituent of MCM2-7 licensing factor complex, was performed to identify the region responsible for its biochemical activities. Partial digestion with trypsin dissected the MCM2 protein into a central region (148-676) containing ATPase motifs and a C-terminal region (677-895). These two fragments, along with three other fragments (148-441, 442-676 and 442-895), were produced using the wheat germ cell-free system and were examined for their ability to inhibit MCM4/6/7 helicase activity. Two fragments (442-895 and 677-895) containing the C-terminus were partly inhibitory to the activity. Further dissection revealed that one fragment (713-895) has strong inhibitory activity. The inhibitory activity of the smaller fragments derived from the C-terminal region correlated with their ability to inhibit SV40 T antigen helicase activity and also with their ability to bind to ssDNA, which has been shown by gel mobility shift analysis. These results strongly suggest that the MCM2 fragments derived from the C-terminal region inhibit DNA helicase activity through their ability to bind to ssDNA. In contrast, two fragments (148-441 and 442-676) from the central region were mainly responsible for the interaction between MCM2 and MCM4, and this was revealed by a pulldown analysis using MCM4 protein beads. Finally, only complete MCM2, not the smaller fragments, could disassemble the MCM4/6/7 hexamer into the MCM2/4/6/7 tetramer.

The aim of this work is to define a simplified semi-analytical beam transportation code that can calculate the spatial distribution of projectile fragments which are widely distributed in a patient's body during heavy-ion beam radiotherapy. In this code, we employed an elemental pencil beam model where the spatial distribution of radiation quality for an elemental beam is calculated and superposed according to the emittance ellipse of the narrow heavy-ion beam determined at the entrance of the target. The radiation quality for an elemental beam was calculated using Goldhaber's model of fragment distribution. The calculation results were compared with the experimental observations for a mono-energetic narrow (12)C beam measured at the secondary beam line in HIMAC. Despite its simplicity, the developed code could reproduce the experimental results well.

For the effective utilization of the favorable properties of heavy-ion beams in cancer treatment, it is important to evaluate the range of incident ions and the deposited dose distribution in a patient's body. For this purpose, the utilization of positron emitters induced in therapeutic irradiation is, at present, the only feasible method for in situ and non-invasive monitoring of heavy-ion therapy. In this study, we performed irradiation experiments with three species of ions to a water target, and the annihilation gamma-ray distributions were obtained with a positron camera. We applied the maximum likelihood estimation (MLE) method for the detected distributions to derive the range of incident ions and the deposited dose distribution in the target. As a result, the ranges were determined with a difference between the MLE range R MLE and the experimental range R exp less than 1.1 mm. The good agreement between the estimated dose distribution with the MLE method and the measured one implies that a deposited dose distribution in a target could be estimated from a detected annihilation gamma-ray distribution.

An efficient computational method to calculate NMR chemical shifts of large biomolecular systems is proposed. The method is based on the fragment molecular orbital (FMO) method combined with the gauge-including atomic orbital (GIAO) and continuous set of gauge transformations (CSGT) methods. It accurately reproduced the conventional ab initio NMR values for a 10-residues peptide. The method was also applied to ubiquitin (76 amino-acid residues), and the calculated chemical shifts agree well with experimentally measured shifts. The proposed method requires a much lower computational cost than the conventional ab initio methods to calculate chemical shifts of large systems.

We have measured deexcitation γ rays in the neutron-rich nuclei of 240U, 246Pu, and 250Cm produced by the (18O, 16O) two-neutron transfer reactions, in coincidence with the 16O particles using Si ΔE−E detectors. The γ rays in these nuclei were identified by selecting the kinetic energies of 16O particles, which correspond to the excitation energies in the residual nuclei below the neutron separation energies. The ground-state bands of 240U, 246Pu, and 250Cm were established up to 12+ states and the K π = 0− octupole band of 240U was established up to 9− states. The systematics of the moments of inertia of the ground-state bands for actinide nuclei shows that the deformed subshell closure at N = 152 is sustained for 96Cm isotopes and that it disappears for 94Pu isotopes.

Nucleocapsid protein of HIV, containing two CCHC-type zinc fingers connected by a linker, is a multi-functional protein involved in many critical steps of the HIV life cycle. Several in vitro investigations demonstrated that the reactivities of the first zinc finger flanked by the linker of HIV-1 NCp7 and HIV-2 NCp8 were essential for binding to viral RNA, however, that of the second zinc finger flanked by the linker of NCp7 was very weak and non-specific, whereas the part of NCp8 called NCp8-f2, interacted strongly and specifically with viral RNA. In this study, the three-dimensional structure of NCp8-f2 was determined for the first time. Furthermore, we established that NCp8-f2 specifically binds to the stem-loop SD in viral RNA, and that the hydrophobic cleft and the basic residues close to the cleft were essential for specific binding to SD. We discuss the functional significance of NCp8-f2 for NCp8 being a multi-functional protein.