G4 Molecules Research Articles

Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.

Coassembly of Gemini Surfactants with Double Hydrophilic Block Polyelectrolytes Leading to Complex Nanoassemblies

The electrostatic coassembly of a double hydrophilic block copolymer poly(2-vinylpyridine)-block-poly(ethylene oxide) (P2VP–PEO) with the gemini surfactant 6,6′-(ethane-1,2-diylbis(oxy))bis(sodium 3-dodecyl benzenesulfonate) (G2) was studied in acidic aqueous solutions by a combination of light scattering, SANS, Cryo-TEM, and ITC. As the critical micelle concentration of gemini surfactants is ∼102 times lower than that of analogous single-tail surfactants, polyelectrolyte chains interact with G2 vesicles instead of individual G2 molecules. The study shows that hydrophobicity, bulkiness, and stiffness of G2 domains formed upon its binding to the P2VP chain affect the coassembly and formation of nanoparticles. The study allows for presentation of a comprehensive outline of the coassembling process and elucidating the structure of the formed nanoparticles.

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

Li+ Local Structure in Li–Tetraglyme Solvate Ionic Liquid Revealed by Neutron Total Scattering Experiments with the 6/7Li Isotopic Substitution Technique

Equimolar mixtures of lithium bis(trifluoromethanesulfonyl)amide (LiTFSA) and tetraglyme (G4: CH3O-(CH2CH2O)4-CH3) yield the solvate (or chelate) ionic liquid [Li(G4)][TFSA], which is a homogeneous transparent solution at room temperature. Solvate ionic liquids (SILs) are currently attracting increasing research interest, especially as new electrolytes for Li-sulfur batteries. Here, we performed neutron total scattering experiments with (6/7)Li isotopic substitution to reveal the Li(+) solvation/local structure in [Li(G4)][TFSA] SILs. The experimental interference function and radial distribution function around Li(+) agree well with predictions from ab initio calculations and MD simulations. The model solvation/local structure was optimized with nonlinear least-squares analysis to yield structural parameters. The refined Li(+) solvation/local structure in the [Li(G4)][TFSA] SIL shows that lithium cations are not coordinated to all five oxygen atoms of the G4 molecule (deficient five-coordination) but only to four of them (actual four-coordination). The solvate cation is thus considerably distorted, which can be ascribed to the limited phase space of the ethylene oxide chain and competition for coordination sites from the TFSA anion.

Thermal and Electrochemical Stability of Tetraglyme–Magnesium Bis(trifluoromethanesulfonyl)amide Complex: Electric Field Effect of Divalent Cation on Solvate Stability

Phase behavior of binary mixtures of tetraglyme (G4) and Mg[TFSA]2 (TFSA: bis(trifluoromethanesulfonyl)amide) was investigated. In a 1:1 molar ratio, G4 and Mg[TFSA]2 formed a stable complex with a melting point of 137 °C. X-ray crystallography of a single crystal of the complex grown from a G4-Mg[TFSA]2 binary mixture revealed that the G4 molecule wraps around Mg2+ to form a complex [Mg(G4)]2+ cation, and the two [TFSA]− anions also participate in the Mg2+ coordination in the crystal. The thermal stability of [Mg(G4)][TFSA]2 was examined by thermogravimetry, and it was found that the complex is stable up to 250 °C. Above 250 °C, desolvation of the Mg2+ ion takes place and G4 evaporates. On the other hand, the weight loss starts at around 140 °C in solutions containing excess G4 (n > 1 in Mg[TFSA]2:G4 = 1:n) due to the evaporation of free (uncoordinated) G4. The suppression of G4 volatility in the [Mg(G4)][TFSA]2 complex is attributed to strong electrostatic and induction interactions between divalent Mg2...

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

Two Dimensional Aggregation Behaviors of Quinoxaline Dendrimers.

This study focuses on the molecular behavior of two dendrimers containing a hydrophilic core group (carboxyl group) and hydrophobic branches (quinoxaline and methoxyphenyl groups), 2,3-bis(4-(2,3- bis(4-methoxyphenyl)quinoxalin-6-yloxy)phenyl)quinoxaline-6-carb-oxylic acid (G2) and 2,3-bis(4-(2,3-bis(4-(2,3-bis(4-methoxyphenyl)quinoxalin-6-yloxy)phe-nyl)quinoxalin-6-y-oxy)phenyl) quin oxaline-6-carboxylic acid (G3) at the air-water interface. To understand the mechanism of the self-assembly of these molecules, we measured the surface pressure-area (III-A) isotherm and investigated the surface morphology of Langmuir-Blodgett films transferred onto hydrophilic silicon wafers using atomic force microscopy (AFM). Upon compression, G2 molecules stand up and steadily make close-packed monolayer whereas G3 molecules form circular domains and gradually make aggregates of domains. These results were confirmed by the X-ray Reflectivity (XRR) profiles of G2 and G3 monolayers transferred onto silicon substrates.

Molecular dynamics simulation on the interaction between single-walled carbon nanotubes and binaphthyl core-based chiral phenylene dendrimers

Abstract

Amide-Based Ionic Liquid and Glyme Mixtures As Halogen-Free Electrolytes for Mg-Secondary Battery

~Introduction~ Magnesium (Mg) is getting a lot more attention as a negative electrode material for a next generation secondary battery because of its high energy density (3839 mAh cm–3), negative electrode potential (–2.356 V vs. SHE) and natural abundance. Although some groups have reported the Mg deposition/dissolution process in ethereal solutions, these electrolytes (e.g. Grignard reagent) are too dangerous to put into practical use due to their high volatility and flammability [1]. In 2013, Abe et al. showed the reversibility of Mg deposition/dissolution in glymes dissolving MgCl2 and AlCl3 [2]. Since glymes have high boiling points and burning points over 100 ˚C, this report promises the practical realization of Mg-secondary battery. Previously reported electrolytes, including Abe’s, for Mg batteries mainly consist of halide compounds where Mg-halogen-ether complexes are electrochemically active. However, halide anions can be anodized to generate halogen gas which have high environmental burden. Recently, we have reported the electrodeposition of Mg from the safe electrolyte for practical use without halide anion at room temperature [3]. This electrolyte contains diglyme (G2) and ionic liquid (IL) n-methyl-n-proplylpiperidinium bis[(trifluoromethyl)sulfonyl]amide (PP13-Tf2N) as solvents, and a simple amide salt Mg(Tf2N)2 as a solute. In previous report, we prepared PP13-Tf2N and G2 mixture 1:4 by volume dissolving 0.1 M Mg(Tf2N)2 (Mg2+ : PP13-Tf2N : G2 = 1:7:56 by mol). However, this mixture mainly consists of glymes (80 volume%); in this report, we investigated electrochemical properties of electrolytes where the fractions of glymes were reduced. ~Experimental~ We mixed the IL with three kinds of glymes (i.e. G2, triglyme (G3), tetraglyme (G4)) at a ratio of Mg2+ : PP13-Tf2N : glymes = 1:7:8 by mol so that every G2 molecules coordinate with Mg2+ and/or PP13+ where lower volatility should be expected. For the electrochemical measurements, Platinum (Pt) sheet and Mg rod (for cyclic voltammetory) / sheet (for garvanostatic electrolysis) was used as working electrode and counter electrode, respectively. Similar to ref. 2, we used Mg immersed in 0.95 mol dm–3 (M) ethylmagnesium bromide / tetrahydrofuran as reference electrode separated from the main electrolytes by porous vycor glass [3]. ~Results & Discussion~ Figure 1 shows the cyclic voltammograms (CVs) measured in these electrolytes. For comparison, Fig. 1a is the previously reported CV for Cu electrode in the G2 mixture at a ratio of Mg2+ : PP13-Tf2N : G2 = 1:7:56 by mol [3]. G2 (Fig. 1b) and G4 (Fig. 1c) mixtures demonstrate the reversible deposition/dissolution process of Mg with large current density and, in 1:7:8 G2 mixture, got larger current density than that Fig. 1a. Although kinematic viscosities of all 1:7:8 mixtures were an order of magnitude higher (25.2—26.0 mm2 s–1) than the case for Fig.1a (1.31 mm2 s–1), the conductivities were comparable (2.59—3.82 mS cm–1) [3]. Therefore, the increase in balk concentration of Mg2+ with remaining high ion conductivities helps to get large current densities. While cathodic overvoltage of Mg deposition was not observed in the 1:7:56 G2 mixture, those in the 1:7:8 mixtures were about 0.2 V, probably due to the change of WE from Cu to Pt. All CVs show the large overvoltages of anodic dissolution, probably because of the formation of surface passivation films. Enhancement of the anodic dissolution process is the challenge for the future. Figure 2 is the photograph and SEM images of Pt electrode after galvanostatic electrolysis in the G4 mixture at 1.5 mA cm–2 for 5685 sec up to 15 C. We obtained flat deposits with metallic luster and it turned out to be elemental Mg by X-ray diffraction. The deposits were not round [3] but angular with a smaller diameter of few micrometers; the SEM images also confirmed that the deposits were not dendritic. ~References~ [1] H. D. Yoo, I. Shterenberg, Y. Gofer, G. Gershinsky, N. Pour, and D. Aurbach, Energy Environ. Sci., 6, 2265 (2013).[2] R. Yasui, K. Asaka, K. Miyazaki, T. Fukutsuka, T. Abe, K. Nishino, and Y. Uchimoto, s of the Electrochemical Society of Japan, 80, 240 (2013).[3] A. Kitada, Y. Kang, Y. Uchimoto, and K. Murase, J. Electrochem. Soc., 161, D102 (2014).

Vibrational spectra study of phosphorus dendrimer containing azobenzene units on the surface

The FTIR and FT Raman spectra of the first generation dendrimers, possessing oxybenzaldehyde (G1) or oxyphenylazobenzaldehyde (G2) terminal groups and sodium 4-[4-oxyphenyl)azo]-benzaldehyde (SOAB) were studied. The structural optimization and normal mode analysis were performed for dendrimer G2 on the basis of the density functional theory (DFT). These calculations gave the frequencies of vibrations, infrared intensities and Raman scattering activities for the E- and Z-forms of azobenzene unit. The energy differences between the E- and Z-forms are 12.62 and 25.16kcal/mol for SOAB and G2. The calculated in gas phase dipole moments for the E- and Z-forms are equal to 20.86, 18.28D (SOAB) and 7.56, 8.88D (G2). The calculated geometrical parameters and harmonic vibrational frequencies are predicted in a good agreement with the experimental data. It was found that dendrimer G2 molecule has a concave lens structure with planar OC6H4CHNN(CH3)PS and OC6H4NNC6H4CHO fragments and slightly non-planar cyclotriphosphazene core. The experimental IR and Raman spectra of dendrimer G2 were interpreted by means of potential energy distributions. Relying on DFT calculations a complete vibrational assignment is proposed. The strong band 1598cm−1 in the IR spectra show marked changes of the optical density in dependence of substituents in the aromatic ring. The differences in the IR and Raman spectra of SOAB and G2 for the E- and Z-forms of azobenzene units were cleared up. During structural isomerization of azobenzene units, redistribution of band intensities appears to a much higher extent than frequency shifts.

Chemical Synthesis of Bacteriophage G4

BackgroundDue to recent leaps forward in DNA synthesis and sequencing technology, DNA manipulation has been extended to the level of whole-genome synthesis. Bacteriophages occupy a special niche in the micro-organic ecosystem and have potential as a tool for therapeutic agent. The purpose of this study was to carry out chemical synthesis of the bacteriophage G4 and the study of its infectivity.Methodology/Principal FindingsFull-sized genomes of bacteriophage G4 molecules were completed from short overlapping synthetic oligonucleotides by direct assembly polymerase chain reaction and ligase chain reaction followed by fusion polymerase chain reaction with flanking primers. Three novel restriction endonuclease sites were introduced to distinguish the synthetic G4 from the wild type. G4 particles were recovered after electroporation into Escherichia coli and were efficient enough to infect another strain. The phage was validated by electron microscope. Specific polymerase chain reaction assay and restriction analyses of the plaques verified the accuracy of the chemical synthetic genomes.ConclusionsOur results showed that the bacteriophage G4 obtained is synthetic rather than a wild type. Our study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application.

Proteomic Analysis of the Plant−Virus Interaction in Cucumber Mosaic Virus (CMV) Resistant Transgenic Tomato

Cucumber mosaic virus (CMV), a member of the Cucumovirus genus, is the causal agent of several plant diseases in a wide range of host species, causing important economic losses in agriculture. Because of the lack of natural resistance genes in most crops, different genetic engineering strategies have been adopted to obtain virus-resistant plants. In a previous study, we described the engineering of transgenic tomato plants expressing a single-chain variable fragment antibody (scFv G4) that are specifically protected from CMV infection. In this work, we characterized the leaf proteome expressed during compatible plant-virus interaction in wild type and transgenic tomato. Protein changes in both inoculated and apical leaves were revealed using two-dimensional gel electrophoresis (2-DE) coupled to differential in gel electrophoresis (DIGE) technology. A total of 2084 spots were detected, and 50 differentially expressed proteins were identified by nanoscale liquid chromatographic-electrospray ionization-ion trap-tandem mass spectrometry (nLC-ESI-IT-MS/MS). The majority of these proteins were related to photosynthesis (38%), primary metabolism (18%), and defense activity (14%) and demonstrated to be actively down regulated by CMV in infected leaves. Moreover, our analysis revealed that asymptomatic apical leaves of transgenic inoculated plants had no protein profile alteration as compared to control wild type uninfected plants demonstrating that virus infection is confined to the inoculated leaves and systemic spread is hindered by the CMV coat protein (CP)-specific scFv G4 molecules. Our work is the first comparative study on compatible plant-virus interactions between engineered immunoprotected and susceptible wild type tomato plants, contributing to the understanding of antibody-mediated disease resistance mechanisms.

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab–Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs.

Self-Assembly of Asymmetric Fan-Shaped Dendrimers with Different Generation Numbers at the Air−Water Interface

Monolayer formation of two dendrimers containing a hydrophilic core group (COOH) and hydrophobic peripheral groups (anthracene and aryl ether tail groups), 4-{10-[4-(3,5-bis-benzyloxy)-phenyl]-anthracen-9-yl}-benzoic acid (G1) and 4-(10-{4-[3,5-bis-(3,5-bis-benzyloxy)-benzyloxy]-phenyl}-anthracen-9-yl)-benzoic acid (G2), were studied. To understand the mechanism of the self-assembly of these molecules, we measured the surface pressure-surface area (Pi- A) isotherm and investigated the surface texture of Langmuir-Blodgett monolayers transferred onto hydrophilic silicon wafers. Both dendrimers form circular domains at the onset point of surface pressure as a result of the difference in hydrophobicity between the core group and the peripheral end group. The core group has a functional group at the end of dendrimer and can be anchored on the water surface. Upon further compression, monolayer of G1 shows a domain of molecules whereas a monolayer of G2 is aligned in the direction of compression at 10 mN/m. At higher surface pressure (20 mN/m), G1 molecules have several aggregates of domains, but G2 molecules maintain their ordering. These results were confirmed by the electron density profile of G1 and G2 monolayers transferred to silicon substrates, as measured by X-ray reflectivity.

Characterization of Variable Regions of Monoclonal Antibodies by Top-Down Mass Spectrometry

A technique for rapid characterization of variable regions of monoclonal antibodies (mAb) is described. Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. In-source fragmentation has the unique advantage of fragmenting all charge states of a protein at the same time and, thus, greatly improves the sensitivity of the fragment ions over a true MS/MS experiment, where a single charge state is isolated and fragmented. In addition, immediate fragmentation of the protein before tertiary structure formation may also facilitate protein fragmentation. This technique has been proved very useful for top-down analysis of large proteins. In-source fragmentation of mAbs generated a series of fragment ions. In addition to some small b and y ions from the light chain and heavy chain in the low m/z region, a series of b ions corresponding to N-terminal 106-120 residues of both heavy chain and light chain were observed. The cleavage sites for these b ions happen to be near the linker regions between the variable domains and the constant domains of these antibodies. These b ions, therefore, correspond to the entire variable region of each chain. Similar results were obtained for all mAbs analyzed, including both immunoglobulin G1 and G2 molecules. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap. Large number of product ions was observed from these b ions. Many of these product ions are internal fragments between the two disulfide-linked cysteine residues. To demonstrate the capability of the technique, several mAbs were force-oxidized by treating with tert-butyl hydroperoxide, followed by mass spectrometric analysis. In-source fragmentation and MS/MS of the variable region b ions clearly identified the locations of the oxidized methionine.

Probing Hydrogen Bonding and Ion−Carbonyl Interactions by Solid-State 17O NMR Spectroscopy: G-Ribbon and G-Quartet

We report solid-state 17O NMR determination of the 17O NMR tensors for the keto carbonyl oxygen (O6) of guanine in two 17O-enriched guanosine derivatives: [6-17O]guanosine (G1) and 2',3',5'-O-triacetyl-[6-17O]guanosine (G2). In G1.2H2O, guanosine molecules form hydrogen-bonded G-ribbons where the guanine bases are linked by O6...H-N2 and N7...H-N7 hydrogen bonds in a zigzag fashion. In addition, the keto carbonyl oxygen O6 is also weakly hydrogen-bonded to two water molecules of hydration. The experimental 17O NMR tensors determined for the two independent molecules in the asymmetric unit of G1.2H2O are: Molecule A, CQ=7.8+/-0.1 MHz, etaQ=0.45+/-0.05, deltaiso=263+/-2, delta11=460+/-5, delta22=360+/-5, delta33=-30+/-5 ppm; Molecule B, CQ=7.7+/-0.1 MHz, etaQ=0.55+/-0.05, deltaiso=250+/-2, delta11=440+/-5, delta22=340+/-5, delta33=-30+/-5 ppm. In G1/K+ gel, guanosine molecules form extensively stacking G-quartets. In each G-quartet, four guanine bases are linked together by four pairs of O6...H-N1 and N7...H-N2 hydrogen bonds in a cyclic fashion. In addition, each O6 atom is simultaneously coordinated to two K+ ions. For G1/K+ gel, the experimental 17O NMR tensors are: CQ=7.2+/-0.1 MHz, etaQ=0.68+/-0.05, deltaiso=232+/-2, delta11=400+/-5, delta22=300+/-5, delta33=-20+/-5 ppm. In the presence of divalent cations such as Sr2+, Ba2+, and Pb2+, G2 molecules form discrete octamers containing two stacking G-quartets and a central metal ion, that is, (G2)4-M2+-(G2)4. In this case, each O6 atom of the G-quartet is coordinated to only one metal ion. For G2/M2+ octamers, the experimental 17O NMR parameters are: Sr2+, CQ=6.8+/-0.1 MHz, etaQ=1.00+/-0.05, deltaiso=232+/-2 ppm; Ba2+, CQ=7.0+/-0.1 MHz, etaQ=0.68+/-0.05, deltaiso=232+/-2 ppm; Pb2+, CQ=7.2+/-0.1 MHz, etaQ=1.00+/-0.05, deltaiso=232+/-2 ppm. We also perform extensive quantum chemical calculations for the 17O NMR tensors in both G-ribbons and G-quartets. Our results demonstrate that the 17O chemical shift tensor and quadrupole coupling tensor are very sensitive to the presence of hydrogen bonding and ion-carbonyl interactions. Furthermore, the effect from ion-carbonyl interactions is several times stronger than that from hydrogen-bonding interactions. Our results establish a basis for using solid-state 17O NMR as a probe in the study of ion binding in G-quadruplex DNA and ion channel proteins.

Crystal Structure of Pullulanase: Evidence for Parallel Binding of Oligosaccharides in the Active Site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.

Elevation of cyclin B1, active cdc2, and HuR in cervical neoplasia with human papillomavirus type 18 infection

Over 30 cervical epitheliotrophic HPV types may lead to altered biological functions that affect the clinical outcome of HPV infection. In order to determine the regulatory mechanism and effect of different HPV subtypes, we performed functional assays on cdc2, cyclinB1 and HuR in human uterine cervical samples. After confirming 22 HPV types among 95 cervical swabs, 10 cervical tissues, and seven established cell lines using a DNA chip, we evaluated the functional activities of G2 molecules assays, that included; western blotting for cyclin B1, cdc2 and phospho-cdc2 (Y15 and T161), immunoprecipitation for cdc2, a nuclear extraction fractional assay, and RT-PCR for cyclin B1. The expression of cyclin B1 was found to be dependent on HPV type, and was particularly overexpressed in high-risk types, whereas cdc2 was ubiquitously expressed irrespective of HPV type. Phospho-cdc2 and cyclin B1, however, were most intense in HPV18 infected cervical samples. Furthermore, the HuR stabilizing factor of the cyclin B1 transcript was upregulated in HPV 18 infected swabs. Moreover, SiHa cell line showed weaker G2 functional activity than the HeLa cell line. This study demonstrates that HPV-18 decreases the fidelity of mitotic checkpoints and increases cdc2-associated histone H1 kinase activity relative to control populations, and further shows that the G2 checkpoint is aberrant by virtue of the stabilization of cyclin B1 mRNA through the upregulation of HuR protein and the functional form of cdc2, especially in cases with HPV 18 infected cervical lesions.

Novel π-Conjugated Nano-Supramolecules Having Fine-Controlled Metal-Assembling Functions

Abstract Dendritic and cyclic polyphenylazomethines (DPA and CPA) were exploited as π-conjugated polymer ligands having novel topological structures. DPAs were synthesized by the convergent method via dehydration in the presence of TiCl4. DPA G4 molecules have a sphere-like structure with a 2.3-nm diameter, and they are regularly assembled without deformation of the molecule on a plate, as observed by means of GPC, molecular modeling, TEM, AFM, and π–A measurements. The stepwise radial complexation in DPAs with SnCl2 was observed as a stepwise shift in the isosbestic point in the UV–vis spectra; this conclusion was further supported by TEM, NMR, and shell-selective reduction (SSR) of the imines. The order of complexation among the shells permits control by the introduction of electron-withdrawing groups to the DPA structure. CPAn-ab and CPAn-aabb (designated as CPAX-Y where X and Y are the degree of polymerization and whether the polymerization was AB- or AABB-type, respectively) was synthesized via dehydration in the presence of TiCl4, TiCl4(THF)2 or p-toluenesulfonic acid (PTS). The unique structures of CPAn-ab and CPAn-aabb: triangle, square, or oblong, and their regular molecular-packing were revealed by NMR, MD calculations, and X-ray crystal analysis. CPAn-aabb shows reversible redox functions by protic acid doping, unlike conventional polyphenylazomethine (PPA).

Preparation and Heck Reaction of Multidentate Carbosilane Films Derived from Focally Functionalized and Allyl-Terminated Dendrons on Hydrogen-Terminated Silicon(111) Surfaces

Multidentate carbosilane films were prepared by thermally induced hydrosilylation of allyl-terminated carbosilane dendrons of generations 0, 1, and 2 (G0-G2) on hydrogen-terminated silicon(111) surfaces. The dendron molecules contain three (G0), nine (G1), and twenty-seven (G2) allyl groups at the periphery, and a bromophenyl functional group at the focal point. The dendron films were characterized by contact-angle goniometry, ellipsometry, Fourier transform infrared spectroscopy in the attenuated total reflection mode, and X-ray photoelectron spectroscopy (XPS). Upon hydroboration of the remaining allyl groups in the films, the percentage of the introduced boron atoms in the films were measured by XPS. The results indicate the presence of roughly 20%, 27%, and 46% of unreacted allyl groups in the G0, G1, and G2 films, respectively. The mechanistic aspects of the chemisorption of these dendron molecules on H-Si(111) surfaces are discussed. XPS studies indicate that seven G0 molecules cover approximately the same area on the substrate as three G1 molecules and one G2 molecule. After treatment of the G0, G1, and G2 films with 4-fluorostyrene under the Heck reaction conditions, the XPS studies indicate that about 84%, 71%, and 55% of the Br atoms were consumed, yielding the replacement of ca. 58-70% of the reacted Br atoms by the fluorostyryl groups. The remaining bromophenyl groups were inactive toward the Heck reaction, probably due to their disfavorable position/orientation in the films.