Modelling plexcitons of periodic gold nanorod arrays with molecular components.

Plasmonic or exciton/plasmon (plexcitonic) systems are presently described based on electromagnetic models, ignoring the need for an improved microscopic understanding. This is based on the fact that a full quantum mechanical approach on a micrometer scale still represents a considerable challenge. In this paper we report on the experimental observation of plexcitons in 2D gold nanorod array systems coupled to dye molecules and we provide a description of the experimental data using a quantum model. We show that treating the collective behavior in the array as being represented by a single quasiparticle is a suitable approximation that offers the opportunity to avoid the complicated calculation of long-distance interactions between the individual nanoparticles of the plexcitonic, periodic system. This enables us to model the optical response of plasmons in nanostructured arrays in contact with quantum emitters and to derive microscopic informations. Our work provides a potential tool for the design of plexcitonic devices, which rely on periodic metallic nanostructures.

Magneto-Stark effect of excitons as the origin of second harmonic generation in ZnO.

The magneto-Stark effect of excitons is demonstrated to be an efficient source of optical nonlinearity in hexagonal ZnO. Strong resonant second harmonic generation signals induced by an external magnetic field are observed in the spectral range of 2s and 2p excitons. The microscopic theoretical analysis shows that for excitons with a finite wave vector, exciton states of opposite parity are mixed by an effective odd parity electric field induced by the magnetic field despite its even parity. The field, spectral, and polarization dependencies of the second harmonic generation intensity validate the proposed mechanism. The observed phenomenon is not limited to a certain symmetry class and therefore must be effective in other semiconductors.

Role of DNA 5-methylcytosine transferase in cell transformation by fos.

The Fos and Jun oncoproteins form dimeric complexes that stimulate transcription of genes containing activator protein-1 regulatory elements. We found, by representational difference analysis, that expression of DNA 5-methylcytosine transferase (dnmt1) in fos-transformed cells is three times the expression in normal fibroblasts and that fos-transformed cells contain about 20 percent more 5-methylcytosine than normal fibroblasts. Transfection of the gene encoding Dnmt1 induced morphological transformation, whereas inhibition of dnmt1 expression or activity resulted in reversion of fos transformation. Inhibition of histone deacetylase, which associates with methylated DNA, also caused reversion. These results suggest that fos may transform cells through alterations in DNA methylation and in histone deacetylation.

Zinc oxide nanorod based photonic devices: recent progress in growth, light emitting diodes and lasers.

Zinc oxide (ZnO), with its excellent luminescent properties and the ease of growth of its nanostructures, holds promise for the development of photonic devices. The recent advances in growth of ZnO nanorods are discussed. Results from both low temperature and high temperature growth approaches are presented. The techniques which are presented include metal-organic chemical vapour deposition (MOCVD), vapour phase epitaxy (VPE), pulse laser deposition (PLD), vapour-liquid-solid (VLS), aqueous chemical growth (ACG) and finally the electrodeposition technique as an example of a selective growth approach. Results from structural as well as optical properties of a variety of ZnO nanorods are shown and analysed using different techniques, including high resolution transmission electron microscopy (HR-TEM), scanning electron microscopy (SEM), photoluminescence (PL) and cathodoluminescence (CL), for both room temperature and for low temperature performance. These results indicate that the grown ZnO nanorods possess reproducible and interesting optical properties. Results on obtaining p-type doping in ZnO micro- and nanorods are also demonstrated using PLD. Three independent indications were found for p-type conducting, phosphorus-doped ZnO nanorods: first, acceptor-related CL peaks, second, opposite transfer characteristics of back-gate field effect transistors using undoped and phosphorus doped wire channels, and finally, rectifying I-V characteristics of ZnO:P nanowire/ZnO:Ga p-n junctions. Then light emitting diodes (LEDs) based on n-ZnO nanorods combined with different technologies (hybrid technologies) are suggested and the recent electrical, as well as electro-optical, characteristics of these LEDs are shown and discussed. The hybrid LEDs reviewed and discussed here are mainly presented for two groups: those based on n-ZnO nanorods and p-type crystalline substrates, and those based on n-ZnO nanorods and p-type amorphous substrates. Promising electroluminescence characteristics aimed at the development of white LEDs are demonstrated. Although some of the presented LEDs show visible emission for applied biases in excess of 10 V, optimized structures are expected to provide the same emission at much lower voltage. Finally, lasing from ZnO nanorods is briefly reviewed. An example of a recent whispering gallery mode (WGM) lasing from ZnO is demonstrated as a way to enhance the stimulated emission from small size structures.

ALK5 promotes tumor angiogenesis by upregulating matrix metalloproteinase-9 in tumor cells.

Transforming growth factor beta 1 (TGF-beta1) is a potent tumor suppressor but, paradoxically, TGF-beta1 enhances tumor growth and metastasis in the late stages of cancer progression. This study investigated the role of TGF-beta type I receptor, ALK5, and three mitogen-activated protein kinases (MAPKs) in metastasis by breast cancer cell line MDA-MB-231. We show that autocrine TGF-beta signaling in MDA-MB-231 cells is required for tumor cell invasion and tumor angiogenesis. Expression of kinase-inactive ALK5 reduces tumor invasion and formation of new blood vessels within the tumor orthotopic xenografts in severe combined immunodeficiency (SCID) mice. In contrast, constitutively active ALK5-T204D enhances tumor invasion and angiogenesis by stimulating expression of matrix metalloproteinase MMP-9/gelatinase-B. Ablation of MMP-9 in ALK5-T204D cells by RNA interference (RNAi) reduces tumor invasion and tumor growth. Importantly, RNAi-MMP-9 reduces tumor neovasculature and increases tumor cell death. Induction of MMP-9 by TGF-beta-ALK5 signaling requires MEK-ERK but not JNK, p38 MAPK or Smad4. Dominant-negative MEK blocks and constitutively active MEK1 enhances MMP-9 expression. However, all three MAPK cascades (ERK, JNK and p38 MAPK) are required for TGF-beta-mediated cell migration. Collectively, our results show that TGF-beta-ALK5-MAPK signaling in tumor cells promotes tumor angiogenesis and MMP-9 is an important component of this program.

Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.

The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.

Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts.

The pseudouridine (psi) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles. Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M. musculus, H. sapiens and Trypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 psi residues. The number of psi residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA of D. melanogaster and M. musculus. Despite this, all of the psi residues were found in three domains, II, IV and V. All three are at or have been linked to the peptidyl transferase center according to the literature. Comparison of the sites for psi among the species examined revealed four conserved or semi-conserved segments. One is the region 1911 to 1917, which contains three psi or modified psi in almost all species examined. This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition. Three additional sites were at the peptidyl transferase center itself. The juxtaposition of the conserved sites for psi with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for psi in ribosome function. We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence of Z. mays chloroplasts and D. melanogaster LSU RNA, correlate the secondary structural context for all known psi residues in ribosomal RNA, and compare the sites for psi with those known for methylated nucleosides in H. sapiens.

Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods.

The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors and acceptors of energy which have been linked to the terminal residues of template polynucleotides (poly- and oligo(U) and oligo(A] and 16 S RNA or to SH-groups of ribosomal proteins S1 and S8. The dynamics of mRNA-ribosome interaction have been investigated by the fluorescence stopped-flow technique. It has been shown that the binding to the 30 S subunit of poly(U) with length much shorter (16 nucleotides) than that covered by the ribosome is greatly enhanced by protein S1. However, the final position of oligo(U)16 on the 30 S subunit, which probably includes the ribosomal decoding site, proves to be quite different from that occupied by oligo(U)16 on a free protein S1. Interaction of oligo- and poly(U) with the 30 S subunit occurs in at least two steps: the first one is as fast as the interaction of poly(U) with free S1, whereas the second step represents a first-order reaction. Therefore, the second step may reflect some rearrangement of the template in the ribosome after its primary binding. It is suggested that protein S1 in some cases may fulfill the role of a transient binding site for mRNA in the course of its interaction with the ribosome. The general shape of the template in the mRNA binding region of the ribosome has been studied using various synthetic ribopolynucleotides and has been shown to be similar. It can be represented by a loop(s) or "U-turn(s)". On the basis of estimation of distances from the ends of poly(U) to some well-localized points on the 30 S ribosomal surface, a tentative model of mRNA path through the ribosome is proposed.

How does the mRNA pass through the ribosome?

A working model of the mRNA path through the ribosome is proposed. According to the model, the template goes around the small ribosomal subunit along the region where its 'head' is separated from other parts of the subunit. The 5'-end of the mRNA fragment covered by the ribosome is located near the 3'-terminus of 16S rRNA, whereas the 3'-terminal residues of the fragment are situated on the outer surface of the subunit, opposite its 'side ledge'. When associated with the 50S subunit, the 30S subunit is oriented in such a manner that the decoding center faces the L7/L12 stalk. Implications of the proposed working model of the mRNA topography for the function of the ribosome are discussed.

Integrin ß5 contributes to the tumorigenic potential of breast cancer cells through the Src-FAK and MEK-ERK signaling pathways.

Cancer progression, response to therapy and metastasis depend on tumor microenvironment. Integrins are cell-adhesion receptors that mediate interactions of cells with extracellular matrix. The αv-ß-family of integrins contributes to tumorigenesis, response to therapy and cancer stem cell biology. Thus, understanding the function of specific integrins in cancer is critical for the development of therapeutic approaches targeting integrins. The study investigated the role of integrin ß5 in breast carcinomas by depleting integrin ß5 using RNA interference and reexpression of integrin ß5. Depletion of integrin ß5 in triple-negative breast carcinoma cells markedly reduced tumor take, growth and tumor angiogenesis, whereas reexpression of integrin ß5 rescued this phenotype. Reduction in tumor angiogenesis is associated with lower expression of vascular endothelial growth factor-A in integrin ß5-depleted tumors. Tumor cells deficient in integrin ß5 have lower migration and proliferative capacities. Biochemical assays revealed that integrin ß5 mediates the Src-focal adhesion kinase and MEK-extracellular signal-regulated kinase signaling events that operate independently, and inhibition of these pathways phenocopies integrin ß5 deficiency. Breast carcinoma cells express high levels of integrin ß5, whereas expression of integrin ß3 is limited to stromal compartments and integrin ß6 is lost in metastatic cells. Together, these findings show a critical role for integrin ß5 in the tumorigenic potential of breast carcinoma cells and therapeutic targeting of integrin ß5 is especially attractive for triple-negative breast carcinomas, which are refractory to most of the current therapies.

TAK1 is required for TGF-beta 1-mediated regulation of matrix metalloproteinase-9 and metastasis.

Transforming growth factor-beta 1 (TGF-beta1) signaling in tumor cells has been implicated in tumor angiogenesis and metastasis by regulating matrix proteolysis. Although MMP-9/gelatinase-B is an important component of these TGF-beta1 responses, the mechanism of its regulation is not well understood. Here, we present evidence that TGF-beta-activated protein kinase 1 (TAK1) is critical for TGF-beta regulation of MMP-9 and the metastatic potential of breast cancer cell line MDA-MB-231. We found that suppression of TAK1 signaling by dominant-negative (dn) TAK1 or RNA interference (siRNA) reduces expression of MMP-9 and tumor cell invasion, without growth inhibition in cell culture. The orthotopic xenograft studies in SCID mice showed that suppression of TAK1 signaling by dn-TAK1 reduces tumor growth and formation of lung metastases. Dn-TAK1 reduced the proliferation Ki-67 index and neovasculature of orthotopic xenografts. TAK1-mediated regulation of MMP-9 involves NF-kappaB signaling. Dn-TAK1 reduces NF-kappaB transcriptional response and inhibition of NF-kappaB reduces expression of MMP-9 and activity of the MMP-9 promoter reporter. Together, these findings suggest that TAK1 contributes to TGF-beta1-mediated tumor angiogenesis and metastasis via a mechanism involving the TAK1-NF-kappaB-MMP-9 pathway.

On the difficulties in characterizing ZnO nanowires.

The electrical properties of single ZnO nanowires grown by vapor phase transport were investigated. While some samples were contacted by Ti/Au electrodes, another set of samples was investigated using a manipulator tip in a low energy electron point-source microscope. The deduced resistivities range from 1 to 10(3) Ωcm. Additionally, the resistivities of nanowires from multiple publications were brought together and compared to the values obtained from our measurements. The overview of all data shows enormous differences (10(-3)-10(5) Ωcm) in the measured resistivities. In order to reveal the origin of the discrepancies, the influence of growth parameters, measuring methods, contact resistances, crystal structures and ambient conditions are investigated and discussed in detail.

Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution.

The number and location of all of the pseudouridine (phi) residues in Saccharomyces cerevisiae small subunit (SSU) ribosomal RNA have been determined by a reverse transcriptase sequencing method [Bakin, A. and Ofengand, J., 1993, Biochemistry, 32, 9754-9762]. Thirteen residues were found in addition to the previously described m1acp3 phi 1189. The residues were scattered throughout the molecule with three being in expansion segments. No phi was found in the three highly conserved single-stranded sequence elements common to all SSU RNAs. Specifically, phi 563, the analog of phi 516 (Escherichia coli) and phi 517 (Bacillus subtilis) were not found. Eight of the phi were located identically to those in mammalian SSU RNA and three were near to mammalian phi residues in the secondary structure. There was no discernible correlation between the sites for phi and the known locations of the methylated nucleosides as exists in large subunit (LSU) RNAs. Comparison of the structural context in which phi was found in SSU RNA with that in LSU RNA showed a differential bias suggestive of possible different roles for phi in the two rRNAs. This work also identified the locations of three putative new modified bases in SSU rRNA, and revealed 15 sequence differences between the yeast strain used here and the reported sequence.

Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique.

A new technique has been developed for the facile location of pseudouridylate (psi) residues in any RNA molecule. The method uses two known modification procedures which in combination uniquely identify U residues which have been converted into psi. The first procedure involves reaction of all U-like and G-like residues with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), followed by alkaline removal of all CMC groups except those linked to the N3 of psi. This stops reverse transcription, resulting in a gel band which identifies the U residue. The second procedure is uridine-specific hydrazinolysis which cleaves the RNA chain at all U residues and produces a gel band upon reverse transcription. psi residues, being resistant to hydrazinolysis, are not cleaved and do not stop reverse transcription. This leads to the absence of a band at psi residues. The combined method can also distinguish psi from 5-methyluridine, 4-thiouridine, uridine-5-oxyacetic acid, and 2-thio-5-methylaminomethyluridine as shown by treating rRNA and tRNA species known to contain these modified bases at defined sites. By this procedure, four new sites for psi in Escherichia coli 23S RNA were discovered, and one was disproven. The four new sites are at positions 2457, 2504, 2580, and 2605. The erroneous site is at position 2555. These four new psi residues, which are all in or within 2-3 residues of the peptidyltransferase ring, are thus in a position to play a functional and/or structural role at the peptidyltransferase center.(ABSTRACT TRUNCATED AT 250 WORDS)

Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes.

Analysis of the high molecular weight RNAs of the larger ribosomal subunit of Saccharomyces cerevisiae cytoplasm and mitochondria by a new method [Bakin, A., & Ofengand, J. (1993) Biochemistry 32, 9754-9762] has for the first time located all of the pseudouridine residues present in these two RNAs. Thirty pseudouridines were found in the cytoplasmic RNA, and one was found in the mitochondrial RNA. The 30 cytoplasmic RNA pseudouridines were clustered in three regions of the RNA known to be at or near the peptidyltransferase center. The single pseudouridine in yeast mitochondrial rRNA at position 2819 was also located at the peptidyltransferase center. The localization of pseudouridines at or near the peptidyltransferase center in both cytoplasmic and mitochondrial ribosomes implies a functional role for pseudouridine in peptide bond formation. A correlation was shown to exist between the locations of the pseudouridines determined in this work and the positions of the methylated nucleotides (both 2'-OCH3 and base-methylated) determined previously by others. In addition, this work has tentatively identified the locations of two previously unknown ribothymidine residues, at positions 955 and 2920 in the cytoplasmic rRNA.

Isolation and properties of Escherichia coli 23S-RNA pseudouridine 1911, 1915, 1917 synthase (RluD).

All nine pseudouridine (psi) residues in Escherichia coli 23S RNA are in or very near the peptidyl transfer centre (PTC) of the ribosome. Five psi synthases catalyze synthesis of these nine psi's. Deletion of the gene for one psi synthase, RluD, which directs synthesis of three closely clustered psi's in the decoding site of the PTC, has a profound negative impact on cell growth. We describe the isolation, without amplification from a cloned coding element, of the triple-site modifying enzyme, RluD, the N-terminal sequence of which has been used to clone and express the corresponding gene, rluD. Unlike "expressed" RluD, which so far has not been shown to modify one (1911) of the three closely clustered sites (1911, 1915, 1917), "natural" RluD modifies all three sites; and unlike another pai synthase, RluA, natural RluD has greatly expanded modifying activity at low Mg concentrations. These properties of the expressed and natural forms of RluD are discussed.

A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).

An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction. The gene was cloned behind a (His)6 leader for affinity purification. Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin. The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi. The enzyme has no dependence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA. Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem. Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation. This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The essentiality of these psi residues can now be assessed by disruption of the synthase gene.