Anharmonicity in a double hydrogen transfer reaction studied in a single porphycene molecule on a Cu(110) surface.

Anharmonicity plays a crucial role in hydrogen transfer reactions in hydrogen-bonding systems, which leads to a peculiar spectral line shape of the hydrogen stretching mode as well as highly complex intra/intermolecular vibrational energy relaxation. Single-molecule study with a well-defined model is necessary to elucidate a fundamental mechanism. Recent low-temperature scanning tunnelling microscopy (STM) experiments revealed that the cis↔cis tautomerization in a single porphycene molecule on Cu(110) at 5 K can be induced by vibrational excitation via an inelastic electron tunnelling process and the N-H(D) stretching mode couples with the tautomerization coordinate [Kumagai et al. Phys. Rev. Lett. 2013, 111, 246101]. Here we discuss a pronounced anharmonicity of the N-H stretching mode observed in the STM action spectra and the conductance spectra. Density functional theory calculations find a strong intermode coupling of the N-H stretching with an in-plane bending mode within porphycene on Cu(110).

Spectroscopic and microscopic investigations of tautomerization in porphycenes: condensed phases, supersonic jets, and single molecule studies.

We describe various experimental approaches that have been used to obtain a detailed understanding of double hydrogen transfer in porphycene, a model system for intramolecular hydrogen bonding and tautomerism. The emerging picture is that of a multidimensional tautomerization coordinate, with several vibrational modes acting as reaction-promoters or inhibitors through anharmonic intermode coupling. Tunnelling processes, coherent in the case of isolated molecules and incoherent in condensed phases, are found to play a major role even at elevated temperatures. Single-molecule spectroscopy studies reveal large fluctuations in hydrogen transfer rates observed over time for the same chromophore. Scanning probe microscopy is employed to directly observe the structure and tautomerization dynamics of single molecules adsorbed on metal surfaces and demonstrates how the interactions of the molecules with atoms of the supporting surface affect their static and dynamic properties: different tautomeric forms are stabilized for molecules depending on the surface structure and the reaction mechanism can also change, from a concerted to a stepwise transfer. The scanning probe microscopy studies prove that tautomerization in single molecules can be induced by different stimuli: heat, electron attachment, light, and force exerted by the microscope's tip. Possible applications utilizing tautomerism are discussed in combination with molecular architectures on surfaces, which could pave the way for the development of single-molecule electronics.

Direct Observation of Photoinduced Tautomerization in Single Molecules at a Metal Surface.

Molecular switches are of fundamental importance in nature, and light is an important stimulus to selectively drive the switching process. However, the local dynamics of a conformational change in these molecules remain far from being completely understood at the single-molecule level. Here, we report the direct observation of photoinduced tautomerization in single porphycene molecules on a Cu(111) surface by using a combination of low-temperature scanning tunneling microscopy and laser excitation in the near-infrared to ultraviolet regime. It is found that the thermodynamically stable trans configuration of porphycene can be converted to the metastable cis configuration in a unidirectional fashion by photoirradiation. The wavelength dependence of the tautomerization cross section exhibits a steep increase around 2 eV and demonstrates that excitation of the Cu d-band electrons and the resulting hot carriers play a dominant role in the photochemical process. Additionally, a pronounced isotope effect in the cross section (∼100) is observed when the transferred hydrogen atoms are substituted with deuterium, indicating a significant contribution of zero-point energy in the reaction. Combined with the study of inelastic tunneling electron-induced tautomerization with the STM, we propose that tautomerization occurs via excitation of molecular vibrations after photoexcitation. Interestingly, the observed cross section of ∼10(-19) cm(2) in the visible-ultraviolet region is much higher than that of previously studied molecular switches on a metal surface, for example, azobenzene derivatives (10(-23)-10(-22) cm(2)). Furthermore, we examined a local environmental impact on the photoinduced tautomerization by varying molecular density on the surface and find substantial changes in the cross section and quenching of the process due to the intermolecular interaction at high density.

Mechanical behavior of nanocrystalline NaCl islands on Cu(111).

The mechanical response of ultrathin NaCl crystallites of nanometer dimensions upon manipulation with the tip of a scanning tunneling microscope (STM) is investigated, expanding STM manipulation to various nanostructuring modes of inorganic materials as cutting, moving, and cracking. In the light of theoretical calculations, our results reveal that atomic-scale NaCl islands can behave elastically and follow a classical Hooke's law. When the elastic limit of the nanocrystallites is reached, the STM tip induces atomic dislocations and consequently the regime of plastic deformation is entered. Our methodology is paving the way to understand the mechanical behavior and properties of other nanoscale materials.

Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

In vitro 32P-labelling of viroid RNA for hybridization studies.

A method is described for the in vitro labelling of viroid RNA for use in hybridization studies. The citrus exocortis viroid (approximately 350 nucleotides) is degraded by hot formamide hydrolysis to fragments ranging from small oligonucleotides to near full lengths, and subsequently labelled to high specific activity by enzymatically attaching 32P to the 5'-end of each molecule. The cleavage step leaves 5' hydroxyl groups which allows the polynucleotide kinase to directly label the RNA fragments without prior enzymatic dephosphorylation. The method is simple, requires no special equipment, and provides a radioactive RNA probe sufficient for most types of hybridization studies.

Melanin production in Escherichia coli from a cloned tyrosinase gene.

We have cloned and functionally expressed a tyrosinase gene from Streptomyces antibioticus in Escherichia coli under the control of an inducible bacteriophage T7 promoter. Recombinant E. coli cells containing the induced tyrosinase gene produced melanin pigments on agar plates and in liquid culture when supplemented with copper and tyrosine. The expression of an additional open reading frame from the mel gene locus of S. antibioticus was required for high-level melanin production in E. coli. Our results also show that it is possible to screen other classes of precursor compounds for incorporation into melanin pigments with unique colors and other biochemical features. In addition, it may be possible to screen for enhanced melanin production in the absence of added precursors to identify overproducing mutants in the amino acid biosynthetic pathways of E. coli. The ability to screen for a melanin phenotype in recombinant E. coli provides new opportunities for production of novel melanins and for protein engineering of tyrosinases with altered catalytic properties.

Conversion of starch to ethanol in a recombinant Saccharomyces cerevisiae strain expressing rice alpha-amylase from a novel Pichia pastoris alcohol oxidase promoter.

A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.

Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus.

Using malaria as a model disease, we engineered the surface of tobacco mosaic tobamovirus (TMV) for presentation of selected epitopes to the mammalian immune system. The TMV coat protein is a well-characterized and abundant self-assembling polymer previously shown to be a highly immunogenic carrier. Selected B-cell epitopes were either inserted into the surface loop region of the TMV coat protein or fused to the C terminus using the leaky stop signal derived from the replicase protein reading frame. Tobacco plants systemically infected with each of these constructs contained high titers of genetically stable recombinant virus, enabling purification of the chimeric particles in high yield. Symptoms induced in tobacco ranged from a normal mosaic pattern similar to that induced by the parental U1 strain to a unique bright yellow mosaic. As measured by quantitative ELISA against synthetic peptide standards, wild type TMV coat protein and fusion protein synthesized by the leaky stop mechanism coassembled into virus particles at the predicted ratio of approximately 20:1. Recombinant plant viruses have the potential to meet the need for scalable and cost effective production of subunit vaccines that can be easily stored and administered.

Controlling on-surface polymerization by hierarchical and substrate-directed growth.

A key challenge in the field of nanotechnology, in particular in the design of molecular machines, novel materials or molecular electronics, is the bottom-up construction of covalently bound molecular architectures in a well-defined arrangement. To date, only rather simple structures have been obtained because of the limitation of one-step connection processes. Indeed, for the formation of sophisticated structures, step-by-step connection of molecules is required. Here, we present a strategy for the covalent connection of molecules in a hierarchical manner by the selective and sequential activation of specific sites, thereby generating species with a programmed reactivity. This approach leads to improved network quality and enables the fabrication of heterogeneous architectures with high selectivity. Furthermore, substrate-directed growth and a preferred orientation of the molecular nanostructures are achieved on an anisotropic surface. The demonstrated control over reactivity and diffusion during covalent bond formation constitutes a promising route towards the creation of sophisticated multi-component molecular nanostructures.

RNA sequences complementary to citrus exocortis viroid in nucleic acid preparations from infected Gynura aurantiaca.

Molecular hybridization with (125)I-labeled citrus exocortis viroid RNA has been used to survey nucleic acid preparations from Gynura aurantiaca for viroid complementary molecules. A differential hybridization effect was detected between nucleic acid extracts from healthy and infected tissue in which significant RNase-resistant (125)I-labeled citrus exocortis viroid resulted in hybridization studies with the infected tissue extracts. Subsequent characterization indicated that RNA from infected tissue was involved in the formation of a duplex molecule with citrus exocortis viroid RNA and had properties of an RNA.RNA hybrid. Subcellular fractionation of infected tissue indicates that the complementary RNA is present in nuclear and soluble RNA fractions. This RNA may represent an intermediate molecule in the replication of the viroid or a pathogenic expression and may have a regulatory role in the host cell.

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba.

The cytoplasmic male sterility (CMS) trait of at least one line of Vicia faba plants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the "447" male sterile line of V.faba plants and could not be isolated from the recurrent parent (maintainer), from lines that had been fertility-restored, or from lines that had reverted from the sterile condition. We have been able to move the CMS-associated RNA from donor to fertile host plants through a dodder bridge. These hots not only contain the RNA but now exhibit a male sterile phenotype, as detected by visual examination of the flower, the pollen, morphological characteristics, and pollen staining ability.

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba.

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient.

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.

Properties of the complementary RNA sequences associated with infection by the citrus exocortis viroid.

The citrus exocortis disease, caused by the low molecular weight viroid RNA, has recently been shown to include viroid complementary RNA in nucleic acid extracts. Purification procedures indicate the presence of viroid complementary sequences in both 2 M LiCl precipitate and 2 M LiCl supernatant fractions. These complementary sequences are detected in preparations from both the high and the low molecular weight regions of polyacrylamide gels. This population heterogeneity coupled with the subcellular distribution indicates viroid-related structures which may be involved in the processes of replication and pathogenesis.

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid.

A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5' ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3' ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.

Synthetic melanin suppresses production of proinflammatory cytokines.

An overproduction of proinflammatory cytokines mediates the damaging sequelae of inflammation in pathologic conditions such as rheumatoid arthritis, graft-vs-host reaction, cachexia, and sepsis syndrome. We examined the cytokine regulatory activity of synthetic melanin, exemplified by biosynthetic l-glycine-l-tyrosine-based polymer (ME-1) and chemosynthetic dihydroxyphenylalanine-based polymer (MC-1). At nontoxic concentrations, both compounds effectively (>/=60%) and reversibly suppressed the production of tumor necrosis factor (TNF), even when applied after stimulation of human peripheral blood monocytes with lipopolysaccharide (LPS). The inhibitory activity of melanin was selective with regard to cytokine response but not inducer- or cell-type-specific. In addition to TNF, melanin inhibited production of interleukin (IL)-1beta, IL-6, and IL-10 but not granulocyte-macrophage colony-stimulating factor by the LPS-stimulated monocytes. Melanin was equally effective in inhibiting production of TNF by monocytes stimulated with the purified protein derivative of Mycobacterium tuberculosis and production of IL-6 by IL-1alpha-stimulated human fibroblasts and endothelial cells. Northern blot analysis, mRNA stability determination, immunoprecipitation studies on metabolically labeled intracellular TNF, and pulse chase experiments revealed that melanin reduced efficiency of mRNA translation. The finding that melanin arrests ongoing cytokine synthesis suggests that this compound may be useful as an adjunct therapy for conditions showing involvement of proinflammatory cytokines.

Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA.

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors.

A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.

[Movement disorders in weightlessness]. / Bewegungsstörungen in der Schwerelosigkeit.

The system MONIMIR has been developed to study the coordination of eye, head and arm movements as well as spinal reflexes in microgravity and was used during three spaceflights on board of the station MIR. The following investigations in the course of the experiment MONIMIR were performed: (1) slow head movements in three planes, (2.3) fast pointing movements of eyes, head and arm to acoustic and visual targets, (4) tracking movements of eyes, head and arm to visual targets, (5) head and arm movements based on short term memory and (6) patellar-tendon-reflex. In microgravity different functions and effectors showed different nature and degree of disturbance and different courses in adaptation; in most of the tests exactness and velocity of head and arm movement was decreased; head movements were more disturbed than arm movements; fast pointing movements were more severely affected than slow tracking movements which partly improved; visual controlled movements showed better adaptation as only proprioceptive controlled movements; the patellar-tendon-reflex was highly increased. Disturbances were most pronounced in the early stage of the spaceflights; at later stages most of the performances improved. Methods and results can be used not only for improvement of election and health control of cosmonauts/astronauts for future longterm space missions but also for diagnostics and research of adaptational processes in course of diseases or extreme conditions on earth.