Toward Cove-Edged Low Band Gap Graphene Nanoribbons

Graphene nanoribbons (GNRs), defined as nanometer-wide strips of graphene, have attracted increasing attention as promising candidates for next-generation semiconductors. Here, we demonstrate a bottom-up strategy toward novel low band gap GNRs (E-g = 1.70 eV) with a well-defined cove-type periphery both in solution and on a solid substrate surface with chrysene as the key monomer. Corresponding cyclized chrysene-based oligornerS consisting of the dimer and tetramer are obtained via an Ullmann Coupling followed by oxidative intramolecular cyclodehydrogenation in solution, and much higher GNR homologues via on-surface synthesis. These oligomers adopt nonplanar structures due to the isteric repulsion between the two C-H bonds at the inner cove position. Characterizations by single crystal X-ray analysis, UV-vis absorption spectroscopy, NMR spectroscopy, and scanning tunneling microscopy (STM) are described. The interpretation is assisted by density functional theory (DFT) calculations.

EXPRESSION OF THE HUMAN PLACENTAL LACTOGEN GENES

The placenta is the site of synthesis of various peptide and steroid hormones related to pregnancy. Human placental lactogen (hPL) is the predominant peptide hormone secreted by term placenta and its synthesis is tissue-specific and coupled to placenta development. The objective of this work was to study the structure and expression of the hPL.^ Poly(A('+))RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to pre-hPL and a minor band comigrating with mature hPL, represent (TURN)15% of the total radioactively labeled proteins. Analysis of the poly(A('+))RNA showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with {('32)P}-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460 and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A('+))RNA was used to construct a cDNA library, of which approximately 5% of the clones were found to hybridize to hPL DNA sequences. Heteroduplexes constructed between a clone containing a 815 bp hPL cDNA insert and a hPL genomic DNA clone revealed four small intervening sequences which can account for the lengths observed in hnRNA molecules.^ Recombinant plasmid HCS-pBR322 containing a 550 bp insert of a cDNA transcript of human placental lactogen (hPL) mRNA was ('3)H-labeled an hybridized in situ to human chromosome preparations. These experiments allowed assignment of the hPL and growth hormone (hGH) genes, which have over 90% nucleotide homology in their coding sequences, to band q22-24 of chromosome 17. A gene copy number experiment showed that both genes are present in (TURN)3 copies per haploid genome.^ Experiments were designed to determine if all members of the hPL gene cluster, consisting of four non-allelic genes, are transcribed in term placenta. Advantage was taken of differences in restriction endonuclease sites in the coding portions of the different hPL genes, to distinguish the putative cDNAs of the transcriptionally active genes. Two genes were found to be represented in the cDNA library and their cDNA transcripts were isolated and characterized. Three independent methods showed that their corresponding mRNAs are about equally represented in the hPL mRNA population. The two cDNAs code for prehPL proteins which differ at a single amino acid position. However the secreted hPLs have identical amino acid sequences. A tetramer insertion duplication was found in a palindrome area of the 3' untranslated region of one of the hPL mRNAs. ^

The Effects of Physical and Chemical Modulating Agents on the Induction of Vaccine-Induced Immune Responses

After the development of the viral-based prostate cancer vaccine, Ad5-PSA, much research has been orientated to help enhance the induced immune response by combining the vaccine with physical and chemical modulating agents, more specifically the polymers polyethylenimine (PEI), chitosan, and chitosan coated with CD3 complex antibodies; all previously shown to stimulate an immune response as isolated gene carriers. To compare the vaccine-induced immune responses between the naked vaccine and the polymer-vaccine combinations, a mouse model using the ovalbumin- specific Ad-OVA vaccine was tested using intracellular cytokine staining (ICS), tetramer staining, and cytotoxic T-cell lymphocyte assays to measure the activation of CD8+ T-cells, interferon gamma proteins (INFƒ×), and the induced cytotoxicity to ovalbumin. The Ad-OVA vaccine combined with both chitosan and chitosan with CD3 complex antibodies, both natural polymers, were found to induce similar immune responses to the naked vaccine while the vaccine combined with the synthetic polymer, PEI, diminished the immune response.

Prime and boost vaccination against HER2/neu in breast cancer

Breast cancer is the most common cancer among women with approximately 180,000 new cases being diagnosed yearly in the United States (1). HER2/neu gene amplification and subsequent protein overexpression is found in 20–30% of breast cancer patients and can lead to the promotion of various metastasis-related properties (2–4) and/or resistance to cancer therapies such as chemotherapy and radiation (5). ^ The protein product of the HER2/neu gene, p185, is a proven target for immunological therapy. Recently, passive immunotherapy with the monoclonal antibody Trastuzumab® has validated an immunological approach to HER2/neu+ breast cancer. Immunity to HER2/ neu, when found in breast cancer patients, is of low magnitude. Vaccination-induced HER2/neu-specific antibodies and HER2/neu-specific cytotoxic T cells could result in long-lived immunity with therapeutic benefit. Many features of DNA vaccines and attenuated viral vectors may contribute to the efficacy of prime-boost vaccination. In particular, vaccines capable of eliciting strong cell-mediated immunity are thought to hold the greatest promise for control of cancer (6–9). ^ To optimize cellular immunization to HER2/neu in my study, the HER2/neu gene was presented to the immune system using a priming vector followed by a second vector used as the boost. In both animals and humans, priming with DNA and boosting with a poxviruses, vaccinia or canarypox appears to be particularly promising for induction of a broad immune responses (10). ^ I tested three gene vaccines encoding the HER2/neu gene: (1) a plasmid, SINCP, that contains part of the genome of Sindbis virus; (2) Viral Replicon Particles (VRP) of Venezuela Equine Encephalitis virus (VEE) and (3) E1/E2a-deleted human Type 5 Adenovirus. In SINCP and the VRP, the caspid and envelope genes of the virus were deleted and replaced with the gene for HER2/neu. SINCP-neu, VRP- neu and Adeno-neu when used alone were effective vaccines protecting healthy mice from challenge with a breast cancer cell line injected in the mammary fat pad or injected i.v. to induce experimental lung metastasis. However, SINCP-neu, VRP-neu or Adeno-neu when used alone were not able to prolong survival of mice in therapeutic models in which vaccination occurred after injection of a breast cancer cell line. ^ When the vaccines were combined in a mixed regimen of a SINCP- neu prime VRP-neu or Adeno-neu boost, there was a significant difference in tumor growth and survival in the therapeutic vaccine models. In vitro assays demonstrated that vaccination with each of the three vaccines induced IgG specific for p185, the gene product of HER2/neu, induced p185-specific T lymphocytes, as measured by tetramer analysis. Vaccination also induced intracellular INF-γ and a positive ELISPOT assay. These findings indicate that SINCP-neu, VRP-neu and Adeno-neu, used alone or in combination, may have clinical potential as adjuvant immunotherapy for the treatment of HER2/neu-expressing tumors. ^

Characterization of the Putative Xyloglucan Glycosyltransferase GT14 in Arabidopsis thaliana

Plant cell walls largely consist of matrix polysaccharides that are linked to cellulose microfibrils. Xyloglucan, the primary hemicellulose of the cell wall matrix, consists of a repeating glucose tetramer structure with xylose residues attached to the first three units ('XXXG'). In Arabidopsis thaliana, the core XXXG structure is further modified by enzymatic addition of galactose and fucose residues to the xylose side chains to produce XLXG, XXLG, XLLG and XLFG structures. GT14 is a putative glycosyltransferase in the GT47 gene family. Initial predictions of GT14's hydrophobic regions, based on its translated amino acid sequence, are almost identical to its Arabidopsis homolog MUR3, which is a xyloglucan galactosyltransferase targeted to the Golgi membrane. This suggests that, like MUR3, GT14 possesses a transmembrane domain and that it is targeted to the Golgi. The monosaccharide composition of leaves from T-DNA insertion knockouts of GT14 was analyzed by gas-liquid chromatography. The gt14 plants were found to have lower fucose and higher mannose contents than wild type plants. Analysis of cell wall and soluble fractions from gt14 and wild type plants revealed that most of the deficiency in fucose was accounted for in the cell wall, supporting the idea that GT14's target is xyloglucan. Finally, gt14 and wild type plants were transformed with GT14 for complementation and overexpression analysis. The majority of transformed plants did not show significant changes with regard to monosaccharide composition. This may be because the plants were in the T1 generation and, thus, hemizygous. Analysis of homozygous plants in the T2 generation may reveal noticeable changes. Further studies on the xyloglucan composition of gt14 plants are necessary to put the observed reduction in cell wall fucose into a meaningful context.

INDUCTION OF STRONGER AND LONG LASTING VACCINE IMMUNITY TO TUBERCULOSIS

Tuberculosis is a major cause of death due to an infection in mankind. BCG vaccine protects against childhood tuberculosis although, it fails to protect against adult tuberculosis. BCG vaccine localizes to immature phagosomes of macrophages, and avoids lysosomal fusion, which decreases peptide antigen production. Peptides are essential for macrophage-mediated priming of CD4 and CD8 T cells respectively through MHC-II and MHC-I pathways. Furthermore, BCG reduces the expression of MHC-II in macrophages of mice after infection, through Toll-like receptor-1/2 (TLR-1/2) mediated signaling. In my first aim, I hypothesized that BCG-induced reduction of MHC-II levels in macrophages can decrease CD4 T cell function, while activation of other surface Toll-like receptors (TLR) can enhance CD4 T cell function. An in vitro antigen presentation model was used where, TLR activated macrophages presented an epitope of Ag85B, a major immunogen of BCG to CD4 T cells, and T cell derived IL-2 was quantitated as a measure of antigen presentation. Macrophages with BCG were poor presenters of Ag85B while, TLR-7/9/5/4 and 1/2 activation led to an enhanced antigen presentation. Furthermore, TLR-7/9 activation was found to down-regulate the degradation of MHC-II through ubiquitin ligase MARCH1, and also stimulate MHC-II expression through activation of AP-1 and CREB transcription elements via p38 and ERK1/2 MAP kinases. I conclude from Aim-I studies that TLR-7/9 ligands can be used as more effective ‘adjuvants’ for BCG vaccine. In Aim-II, I evaluated the poor CD8 T cell function in BCG vaccinated mice thought to be due to a decreased leak of antigens into cytosol from immature phagosomes, which reduces the MHC-I mediated activation of CD8 T cells. I hypothesized that rapamycin co-treatment could boost CD8 T cell function since it was known to sort BCG vaccine into lysosomes increasing peptide generation, and it also enhanced the longevity of CD8 T cells. Since CD8 T cell function is a dynamic event better measurable in vivo, mice were given BCG vaccine with or without rapamycin injections and challenged with virulent Mycobacterium tuberculosis. Organs were analysed for tetramer or surface marker stained CD8 T cells using flow cytometry, and bacterial counts of organisms for evaluation of BCG-induced protection. Co-administration of rapamycin with BCG significantly increased the numbers of CD8 T cells in mice which developed into both short living effector- SLEC type of CD8 T cells, and memory precursor effector-MPEC type of longer-living CD8 T cells. Increased levels of tetramer specific-CD8 T cells correlated with a better protection against tuberculosis in rapamycin-BCG group compared to BCG vaccinated mice. When rapamycin-BCG mice were rested and re-challenged with M.tuberculosis, MPECs underwent stronger recall expansion and protected better against re-infection than mice vaccinated with BCG alone. Since BCG induced immunity wanes with time in humans, we made two novel observations in this study that adjuvant activation of BCG vaccine and rapamycin co-treatment both lead to a stronger and longer vaccine-mediated immunity to tuberculosis.

Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization

We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.

Human argininosuccinate lyase: A structural basis for intragenic complementation

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits enzymatic activity that is greater than that found in the oligomeric proteins produced by each mutant allele alone. The mutations involved in the most successful complementation event observed in ASL deficiency were found to be an aspartate to glycine mutation at codon 87 of one allele (D87G) coupled with a glutamine to arginine mutation at codon 286 of the other (Q286R). To understand the structural basis of the Q286R:D87G intragenic complementation event at the ASL locus, we have determined the x-ray crystal structure of recombinant human ASL at 4.0 Å resolution. The structure has been refined to an R factor of 18.8%. Two monomers related by a noncrystallographic 2-fold axis comprise the asymmetric unit, and a crystallographic 2-fold axis of space group P3121 completes the tetramer. Each of the four active sites is composed of residues from three monomers. Structural mapping of the Q286R and D87G mutations indicate that both are near the active site and each is contributed by a different monomer. Thus when mutant monomers combine randomly such that one active site contains both mutations, it is required by molecular symmetry that another active site exists with no mutations. These “native” active sites give rise to the observed partial recovery of enzymatic activity.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-d-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42°C. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels

Native cylic nucleotide-gated (CNG) channels are composed of α and β subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the α and β subunits. Dimers contained three subunit types: αwt, βwt, and αm. The αm subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of αwtαwt dimers and from αwt monomers were indistinguishable. Channels from injection of αwtαm dimers had apparent affinities 3-fold lower than αwt homomultimers, suggesting a channel with two αwt and two αm subunits. Channels from coinjection of αwtαwt and ββ dimers were indistinguishable from those composed of α and β monomers and shared all of the characteristics of the α+β phenotype of heteromeric channels. Coinjection of αwtαm and ββ dimers yielded channels also of the α+β phenotype but with an apparent affinity 3-fold lower, indicating the presence of αm in the tetramer and that α+β channels have adjacent α-subunits. To distinguish between an α-α-α-β and an α-α-β-β arrangement, we compared apparent affinities for channels from coinjection of αwtαwt and βαwt or αwtαwt and βαm dimers. These channels were indistinguishable. To further argue against an α-α-α-β arrangement, we quantitatively compared dose–response data for channels from coinjection of αwtαm and ββ dimers to those from α and β monomers. Taken together, our results are most consistent with an α-α-β-β arrangement for the heteromeric olfactory CNG channel.

Archaeal nucleosomes

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect ≈60 bp of DNA and multiples of ≈60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.

Peptide interfacial adsorption is kinetically limited by the thermodynamic stability of self association

We present a study of the adsorption of two peptides at the octane–water interface. The first peptide, Lac21, exists in mixed monomer–tetramer equilibrium in bulk solution with an appreciable monomer concentration. The second peptide, Lac28, exists as a tetramer in solution, with minimal exposed hydrophobic surface. A kinetic limitation to interfacial adsorption exists for Lac28 at moderate to high surface coverage that is not observed for Lac21. We estimate the potential energy barrier for Lac28 adsorption to be 42 kJ/mol and show that this is comparable to the expected free energy barrier for tetramer dissociation. This finding suggests that, at moderate to high surface coverage, adsorption is kinetically limited by the availability of interfacially active monomeric “domains” in the subinterfacial region. We also show how the commonly used empirical equation for protein adsorption dynamics can be used to estimate the potential energy barrier for adsorption. Such an approach is shown to be consistent with a formal description of diffusion–adsorption, provided a large potential energy barrier exists. This work demonstrates that the dynamics of interfacial adsorption depend on protein thermodynamic stability, and hence structure, in a quantifiable way.

Inhibiting transthyretin amyloid fibril formation via protein stabilization

Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 μM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis—i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.

Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α1β1 interface, and all of the others are at the sliding α1β2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199–203].

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer–dimer interface of the tetrameric oligomerization domain of p53 (residues 325–355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer–dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.