Probing Electron Spin Resonance in Monolayer Graphene.

The precise value of the g factor in graphene is of fundamental interest for all spin-related properties and their application. We investigate monolayer graphene on a Si/SiO_{2} substrate by resistively detected electron spin resonance. Surprisingly, the magnetic moment and corresponding g factor of 1.952±0.002 is insensitive to charge carrier type, concentration, and mobility.

Two-center interferences in dielectronic transitions in H2(+) + He collisions.

Molecular two-center interferences in a collision induced excitation of H2(+) projectile ions, with simultaneous ionization of helium target atoms, are investigated in a kinematically complete experiment. In the process under investigation, the helium atom is singly ionized and simultaneously the molecular hydrogen ion is dissociated. Different collision mechanisms are identified and interference fringes emerging from a correlated first-order mechanism and from an independent second-order process are observed.

Strong molecular alignment dependence of H2 electron impact ionization dynamics.

Low-energy (E(0) = 54 eV) electron impact single ionization of molecular hydrogen (H(2)) has been investigated as a function of molecular alignment in order to benchmark recent theoretical predictions [Colgan et al., Phys. Rev. Lett. 101, 233201 (2008) and Al-Hagan et al., Nature Phys. 5, 59 (2009)]. In contrast to any previous work, we observe distinct alignment dependence of the (e,2e) cross sections in the perpendicular plane in good overall agreement with results from time-dependent close-coupling calculations. The cross section behavior can be consistently explained by a rescattering of the ejected electron in the molecular potential resulting in an effective focusing along the molecular axis.

Low energy (e, 2e) study from the 1t(2) orbital of CH4.

Single ionization of the methane (CH(4)) 1t(2) orbital by 54 eV electron impact has been studied experimentally and theoretically. The measured triple differential cross sections cover nearly a 4π solid angle for the emission of low energy electrons and a range of projectile scattering angles. Experimental data are compared with theoretical calculations from the distorted wave Born approximation and the molecular three-body distorted wave models. It is found that theory can give a proper description of the main features of experimental cross section only at smaller scattering angles. For larger scattering angles, significant discrepancies between experiment and theory are observed. The importance of the strength of nuclear scattering from the H-nuclei was theoretically tested by reducing the distance between the carbon nuclei and the hydrogen nuclei and improved agreement with experiment was found for both the scattering plane and the perpendicular plane.

Phylogeny, genetic variability and colour polymorphism of an emerging animal model: the short-lived annual Nothobranchius fishes from southern Mozambique.

Nothobranchius are a group of small, extremely short-lived killifishes living in temporary savannah pools in Eastern Africa and that survive annual desiccation of their habitat as dormant eggs encased in dry mud. One mitochondrial (COI) and three nuclear (CX32.2, GHITM, PNP) loci were used to investigate the phylogenetic relationship of Nothobranchius species from southern and central Mozambique. This group shows marked variation in captive lifespan at both the inter- and intraspecific levels; lifespan varies from a few months to over a year. As their distribution encompasses a steep gradient between semi-arid and humid habitats, resulting in contrasting selection pressures on evolution of lifespan and associated life history traits, Mozambican Nothobranchius spp. have recently become a model group in studies of ageing, age-related disorders and life history evolution. Consequently, intraspecific genetic variation and male colour morph distribution was also examined in the recovered clades. Using Bayesian species tree reconstruction and single loci analyses, three large clades were apparent and their phylogenetic substructure was revealed at the inter- and intra-specific levels within those clades. The Nothobranchius furzeri and Nothobranchius orthonotus clades were strongly geographically structured. Further, it was demonstrated that male colour has no phylogenetic signal in N. furzeri, where colour morphs are sympatric, but is associated with two reciprocally monophyletic groups in Nothobranchius rachovii clade, where colour morphs are parapatric. Finally, our analysis showed that a polymorphism in the Melanocortin1 receptor gene (which controls pigmentation in many vertebrates and was a candidate gene of male colouration in N. furzeri) is unrelated to colour phenotypes of the study species. Our results raise significant implications for future comparative studies of the species and populations analysed in the present work.

Reaction microscope for investigating ionization dynamics of weakly bound alkali dimers.

We report on the implementation of a far-off-resonant, optical dipole force trap in a reaction microscope combined with a magneto-optical trap. Kinematically complete multi-photon ionization experiments were performed on optically trapped 6Li atoms and photo-associated 6Li2 molecules in their highest vibrational state. The apparatus allows us to distinguish different ionization mechanisms related to the presence of the IR field of the optical dipole trap that can occur during ionization of 6Li and 6Li2 in strong fields. In a series of proof-of-principle experiments, we detect weakly bound dimers via three-photon ionization with femtosecond pulses (τ = 30 fs) at a central wavelength of 780 nm and measure directly the momenta of the photoelectrons in coincidence with recoil ions.

Identification and further characterization of the specific cell binding fragment from sponge aggregation factor.

Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.

Stress Response and Virulence Potential Modulating Effect of Peppermint Essential Oil in Campylobacter jejuni.

Campylobacter jejuni is one of the most common food-borne bacteria that causes gastrointestinal symptoms. In the present study we have investigated the molecular basis of the anti-Campylobacter effect of peppermint essential oil (PEO), one of the oldest EO used to treat gastrointestinal diseases. Transcriptomic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and proteomic, two-dimensional polyacryl amid gel electrophoresis (2D-PAGE) methods have revealed that, in the presence of a sublethal concentration of PEO, the expression of several virulence-associated genes was decreased (cheY 0.84x; flhB 0.79x; flgE 0.205x; cadF 0.08x; wlaB 0.89x; porA 0.25x; cbf2 4.3x) while impaired motility was revealed with a functional analysis. Scanning electron micrographs of the exposed cells showed that, unlike in the presence of other stresses, the originally curved C. jejuni cells straightened upon PEO exposure. Gaining insight into the molecular background of this stress response, we have revealed that in the presence of PEO C. jejuni dominantly exerts a general stress response that elevates the expression of general stress genes like dnaK, groEL, groES (10.41x, 3.63x, and 4.77x). The most important genes dps, sodB, and katA involved in oxidative stress responses showed however moderate transcriptional elevations (1,58x, 1,55x, and 1,85x).

A subnanosecond pulsed ion source for micrometer focused ion beams.

A new, compact design of an ion source delivers nanosecond pulsed ion beams with low emittance, which can be focused to micrometer size. By using a high-power, 25 fs laser pulse focused into a gas region of 10(-6) mbar, ions at very low temperatures are produced in the small laser focal volume of 5 mum diameter by 20 mum length through multiphoton ionization. These ions are created in a cold environment, not in a hot plasma, and, since the ionization process itself does not significantly heat them, have as a result essentially room temperature. The generated ion pulse, up to several thousand ions per pulse, is extracted from the source volume with ion optical elements that have been carefully designed by simulation calculations. Externally triggered, its subnanosecond duration and even smaller time jitter allow it to be superimposed with other pulsed particle or laser beams. It therefore can be combined with any type of collision experiment where the size and the time structure of the projectile beam crucially affect the achievable experimental resolution.

New B-lymphocyte-specific enhancer-binding protein.

We report the discovery of a new B-lymphocyte-specific enhancer-binding protein. A series of gel retardation assays using fragments that scan the -2172 to -1180 region of the major histocompatibility complex class II gene E alpha reveal a site (W) that serves as the recognition sequence for two nuclear proteins, one B-cell restricted and the other ubiquitously occurring. Certain characteristics of the NF-W1 and NF-W2 pair recall the OTF-2/NF-A2 and OTF-1/NF-A1 pair that binds to the immunoglobulin octamer, but we demonstrate that the two protein pairs are distinguishable by several criteria. NF-W1 and NF-W2 interact differentially with their common GTTGCATC binding site, display a different affinity for it, and have molecular weights that differ by about 20,000. Yet, proteolysis experiments and cross-linking analyses indicate that the two W complexes show structural relatedness.

B-cell control region at the 5' end of a major histocompatibility complex class II gene: sequences and factors.

Transcription of major histocompatibility complex class II genes is elaborately regulated. Mouse class II genes are transcribed primarily in B cells, peripheral macrophages and interdigitating cells, and thymic cortical and medullary cells. In this study, we began to identify the DNA sequences and protein factors that control expression of a class II gene in B cells, addressing in particular how closely they resemble those that regulate immunoglobulin gene expression. We describe a region upstream of the E alpha gene that is crucial for its transcription in the B cells of transgenic mice but is less important in cultured B-cell lines. The sequence of this region reveals several familiar motifs, including a second X-Y pair reminiscent of that residing in the promoter-proximal region of all class II genes, a B motif strikingly homologous to that associated with the immunoglobulin kappa gene enhancer, several Ephrussi motifs, and a Pu box-like sequence very similar to that implicated in simian virus 40 and lymphotrophic papovavirus expression in B cells. Careful study of the proteins that bind specifically to these different motifs prompts us to suggest that major histocompatibility complex class II and immunoglobulin genes rely on quite different factors to achieve B-cell-specific expression.

Characterization of chloroquine-hematin mu-oxo dimer binding by isothermal titration calorimetry.

Numerous studies indicate that a key feature of chloroquine's (CQ) antimalarial activity is its interaction with hematin. We now characterize this CQ-hematin interaction in detail using isothermal titration calorimetry (ITC). Between pH 5.6 and 9.0, association constants (K(a) values) for enthalpy-driven CQ-hematin mu-oxo dimer binding fell in the narrow range of 2.3-4.4 x 10(5) M(-1). It is therefore probable that CQ-hematin mu-oxo dimer binding affinity does not diminish at the pH range (4.8-5.4) of the parasite food vacuole. The binding affinity was unaffected by high salt concentrations, suggesting that ionic interactions do not contribute significantly to this complexation. With increasing ionic strength, the entropic penalty of CQ-hematin mu-oxo dimer binding decreased accompanied by increased hematin mu-oxo dimer aggregation. A stoichiometry (n) of 1:4 in the pH range 6.5-9.0 indicates that CQ binds to two hematin mu-oxo dimers. At pH 5.6, a stoichiometry of 1:8 suggests that CQ binds to an aggregate of four hematin mu-oxo dimers. This work adds further evidence supporting the hypothesis that CQ impedes hematin monomer incorporation into hemozoin by producing a forward shift in the hematin monomer-hematin mu-oxo dimer equilibrium, contributing to a destructive accumulation of soluble forms of hematin in the parasite and leading to its death by hematin poisoning.

Homeodomain proteins in development and therapy.

Homeobox genes encode transcriptional regulators found in all organisms ranging from yeast to humans. In Drosophila, a specific class of homeobox genes, the homeotic genes, specifies the identity of certain spatial units of development. Their genomic organization, in Drosophila, as well as in vertebrates, is uniquely connected with their expression which follows a 5'-posterior-3'-anterior rule along the longitudinal body axis. The 180-bp homeobox is part of the coding sequence of these genes, and the sequence of 60 amino acids it encodes is referred to as the homeodomain. Structural analyses have shown that homeodomains consist of a helix-turn-helix motif that binds the DNA by inserting the recognition helix into the major groove of the DNA and its amino-terminal arm into the adjacent minor groove. Developmental as well as gene regulatory functions of homeobox genes are discussed, with special emphasis on one group, the Antennapedia (Antp) class homeobox genes and a representative 60-amino acid Antennapedia peptide (pAntp). In cultured neuronal cells, pAntp translocates through the membrane specifically and efficiently and accumulates in the nucleus. The internalization process is followed by a strong induction of neuronal morphological differentiation, which raises the possibility that motoneuron growth is controlled by homeodomain proteins. It has been demonstrated that chimeric peptide molecules encompassing pAntp are also captured by cultured neurons and conveyed to their nuclei. This may be of enormous interest for the internalization of drugs.

Tumorigenesis by adenovirus type 12 in newborn Syrian hamsters.

Ad12 oncogenesis in hamsters has been studied in detail to provide the following new data in this tumor model. Cells in the Ad12-induced tumors, often thought to be of neuronal origin, actually exhibit mesenchymal and neuronal characteristics and are probably of an undifferentiated derivation. Their intraperitoneal spread upon intramuscular injection of Ad12 adds another important new aspect. Differences in the integration patterns among the tumors suggest clonal origins from individual transformation events. Ad12 gene expression in the tumors is determined, at least in part, by the patterns of DNA methylation imprinted de novo upon the integrated Ad12 genomes. Differential Ad12 gene expression patterns, which have previously not been described in tumors, are an important parameter in Ad12 oncogenesis. The availability of cellular DNA arrays has opened up unprecedented possibilities to document changes in cellular transcription patterns, particularly of cancer-specific genes. These patterns exhibit differences and similarities among the different Ad12-induced tumors. Among the cellular genes, which are expressed in the Ad12-induced tumors, many are cancer-specific. We pursue the hypothesis that these alterations in cellular transcription patterns as a consequence of viral DNA integration and expression play an essential role in Ad12 oncogenesis.

Renin inhibition by substituted piperidines: a novel paradigm for the inhibition of monomeric aspartic proteinases?

BACKGROUND: The aspartic proteinase renin catalyses the first and rate-limiting step in the conversion of angiotensinogen to the hormone angiotensin II, and therefore plays an important physiological role in the regulation of blood pressure. Numerous potent peptidomimetic inhibitors of this important drug target have been developed, but none of these compounds have progressed past clinical phase II trials. Limited oral bioavailability or excessive production costs have prevented these inhibitors from becoming new antihypertensive drugs. We were interested in developing new nonpeptidomimetic renin inhibitors. RESULTS: High-throughput screening of the Roche compound library identified a simple 3, 4-disubstituted piperidine lead compound. We determined the crystal structures of recombinant human renin complexed with two representatives of this new class. Binding of these substituted piperidine derivatives is accompanied by major induced-fit adaptations around the enzyme's active site. CONCLUSIONS: The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.

The enhancer in an MHC class II gene, in vitro and in mouso.

The E alpha class II gene of the major histocompatibility complex is expressed in a variety of immunocompetent cells. Part of the control of tissue-specific expression is mediated by a block of sequences found far upstream of the transcriptional startsite; this stretch is necessary for expression in the B lymphocytes of transgenic mice, but largely dispensable elsewhere. We review the evidence for the role of this region in E alpha transcription in transgenic animals, as well as data from transfections into tissue-culture cells, which indicate that this region has non-specific enhancer activity. We discuss possible models to explain how a non-specific enhancer can participate in cell-specific control.

Bisquinolines. 2. Antimalarial N,N-bis(7-chloroquinolin-4-yl)heteroalkanediamines.

N,N-Bis(7-chloroquinolin-4-yl)heteroalkanediamines 1-11 were synthesized and screened against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. These bisquinolines had IC50 values from 1 to 100 nM against P. falciparum in vitro. Six of the 11 bisquinolines were significantly more potent against the chloroquine-resistant W2 clone compared to the chloroquine-sensitive D6 clone. For bisquinolines 1-11 there was no relationship between the length of the bisquinoline heteroalkane bridge and antimalarial activity and no correlation between in vitro and in vivo antimalarial activities. Bisquinolines with alkyl ether and piperazine bridges were substantially more effective than bisquinolines with alkylamine bridges against P. berghei in vivo. Bisquinolines 1-10 were potent inhibitors of hematin polymerization with IC50 values falling in the narrow range of 5-20 microM, and there was a correlation between potency of inhibition of hematin polymerization and inhibition of parasite growth. Compared to alkane-bridged bisquinolines (Vennerstrom et al., 1992), none of these heteroalkane-bridged bisquinolines had sufficient antimalarial activity to warrant further investigation of the series.

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Sponge secondary metabolites: biochemical and ultrastructural localization of the antimitotic agent avarol in Dysidea avara.

The secondary metabolite avarol, a potent cytostatic and antibacterial sesquiterpenoid hydroquinone, is present in large amounts only in the sponge Dysidea avara (2.7 g avarol/1 kg of fresh material). The present study was designed to determine the storage site of this compound within the organism. Light and transmission electron microscopic studies revealed that avarol is probably stored only in spherular cells. The compound is compartmented in intracellular cytoplasmic vesicles in a paracrystalline form, and therefore can have no inhibitory effect on the sponge cells. Quantitative analysis utilizing high-pressure liquid chromatography revealed that avarol is present at a concentration of 3.2 micrograms/10(6) spherular cells. It appears that avarol is released from the cells into the extracellular space in a merocrine manner. We suggest that it is involved in regulating the bacteria with which the sponge is symbiotically associated.

Deferoxamine: stimulation of hematin polymerization and antagonism of its inhibition by chloroquine.

The iron chelator deferoxamine enhances the clearance of Plasmodium falciparum parasitemia and may be useful in drug combinations for the treatment of cerebral malaria. However, the deferoxamine-chloroquine drug combination is antagonistic, or at best additive, against P. falciparum in vitro. As chloroquine is thought to exert its antimalarial activity by interacting with hematin released from the proteolytic degradation of hemoglobin in the parasite food vacuole, we hypothesized that deferoxamine might interfere with the ability of chloroquine to inhibit hematin polymerization, since it was reported that deferoxamine interacts with hematin. Therefore, we assessed deferoxamine-hematin binding in more detail and investigated the effect of deferoxamine on hematin polymerization in the presence and absence of chloroquine. Isothermal titration calorimetry (ITC) experiments demonstrated an enthalpy-driven deferoxamine:hematin mu-oxo dimer binding with an association constant of 2.8 x 10(4) M(-1) at pH 6.5, a binding affinity 14-fold lower than that measured for chloroquine. At least two of the three hydroxamic acid functional groups of deferoxamine must be unionized for effective binding. We also discovered that deferoxamine antagonized chloroquine-mediated inhibition of hematin polymerization. Unexpectedly, deferoxamine increased the concentration of soluble forms of hematin and enhanced the rate of hematin polymerization. Deferoxamine also could initiate hematin polymerization. In contrast, chloroquine decreased the concentration of soluble forms of hematin and inhibited hematin polymerization. This work supports the postulate that initiation of hematin polymerization requires a higher concentration of soluble hematin monomer than does the elongation phase of polymerization and provides one possible explanation for the observed antagonism between deferoxamine and chloroquine against parasites in culture.