Electron-Transfer Properties of Phenyleneethynylene Linkers Bound to Gold via a Self-Assembled Monolayer of Molecular Tripod.

The three-point adsorption of tripod-shaped molecules enables the formation of robust self-assembled monolayers (SAMs) on solid surfaces, where the component molecules are fixed in a strictly upright orientation. In the present study, SAMs of a rigid molecular tripod consisting of an adamantane core and three CH2SH groups were employed to arrange ferrocene on a gold surface through oligo(p-phenyleneethynylene) linkers. Cyclic voltammetry of the monolayers demonstrated high surface coverage of ferrocene, yet the molecular interaction among adjacent ferrocene units was negligible. This was because of the extended intermolecular distance caused by the bulky tripod framework. The rates of electron transfer from the ferrocene to the gold surface through different linker lengths were determined by electrochemical measurements, from which the decay factor for oligo(p-phenyleneethynylene) wire was evaluated.

Diphenylcarbene Protected by Four ortho-Iodine Groups: An Unusually Persistent Triplet Carbene.

Diphenyldiazomethane with four iodine groups at the ortho positions and two tert-butyl groups at the para positions, i.e., bis(4-tert-butyl-2,6-diiodophenyl)diazomethane (1a-N2), was synthesized as a sterically hindered triplet carbene precursor. Irradiation of 1a-N2 in solution effectively generated the corresponding triplet diphenylcarbene ³1a, which was characterized by UV-vis spectroscopy at low temperature, along with laser flash photolysis techniques at room temperature. The UV-vis spectrum of ³1a was obtained by irradiating 1a-N2 in a 2-methyltetrahydrofuran matrix at 77 K. The ESR spectrum showed no triplet carbene signals, while a radical species was observed at the anticipated temperature of the decomposition of triplet carbene ³1a. Transient absorption bands ascribable to ³1a were observed by laser flash photolysis of 1a-N2 in a degassed benzene solution and decayed very slowly with a second-order rate constant (2k/εl) of 5.5 × 10-³·s-¹. Steady-state irradiation of 1a-N2 in degassed benzene afforded 9,10-diarylphenanthrene derivative 2a in a 31% yield. Triplet carbene ³1a was also trapped by either oxygen (kO2 = 6.5 × 105 M-¹·s-¹) or 1,4-cyclohexadiene (kCHD = 1.5 M-¹·s-¹) to afford the corresponding ketone 1a-O or the diarylmethane 1a-H2. The carbene was shown to be much less reactive than the triplet diphenylcarbene that is protected by two ortho-iodo and two ortho-bromo groups, ³1b.

Ideal redox behavior of the high-density self-assembled monolayer of a molecular tripod on a Au(111) surface with a terminal ferrocene group.

A dyad consisting of a tripod-shaped trithiol with an adamantane core and a terminal ferrocenyl group linked through ap-phenyleneethynylene bridge was synthesized. The trithiol formed a stable self-assembled monolayer (SAM) on Au(111), wherein each molecule is bound to the surface by three-point adsorption using all sulfur atoms, with confirmation by PM-IRRAS and XPS analyses. Cyclic voltammetry of the SAM showed a line shape typical of an ideal adsorbed system, that is, a monolayer with negligible electrostatic interaction among the terminal ferrocenyl groups. Thus, a rare SAM was achieved, in which the component molecules were isolated from adjacent molecules without the coadsorption of nonelectroactive molecules.

Silencing of WIPK and SIPK mitogen-activated protein kinases reduces tobacco mosaic virus accumulation but permits systemic viral movement in tobacco possessing the N resistance gene.

Infection of tobacco cultivars possessing the N resistance gene with Tobacco mosaic virus (TMV) results in confinement of the virus by necrotic lesions at the infection site. Although the mitogen-activated protein kinases WIPK and SIPK have been implicated in TMV resistance, evidence linking them directly to disease resistance is, as yet, insufficient. Viral multiplication was reduced slightly in WIPK- or SIPK-silenced plants but substantially in WIPK/SIPK-silenced plants, and was correlated with an increase in salicylic acid (SA) and a decrease in jasmonic acid (JA). Silencing of WIPK and SIPK in a tobacco cultivar lacking the N gene did not inhibit viral accumulation. The reduction in viral accumulation was attenuated by expressing a gene for an SA-degrading enzyme or by exogenously applying JA. Inoculation of lower leaves resulted in the systemic spread of TMV and formation of necrotic lesions in uninoculated upper leaves. These results suggested that WIPK and SIPK function to negatively regulate local resistance to TMV accumulation, partially through modulating accumulation of SA and JA in an N-dependent manner, but positively regulate systemic resistance.

Antiviral RNA silencing is restricted to the marginal region of the dark green tissue in the mosaic leaves of tomato mosaic virus-infected tobacco plants.

Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue ( approximately 0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.

Formation of a stannylstannylene via intramolecular carbene addition of a transient stannaacetylene (RSn[triple bond, length as m-dash]CR').

Photolysis of diazomethylstannylene [, Me(3)SiC([double bond, length as m-dash]N(2))(Ar)Sn:, Ar = C(6)H(3)-2,6-Tip(2), Tip = C(6)H(2)-2,4,6-(i-Pr)(3)] afforded unusual stannylstannylene via intramolecular carbene addition to an aromatic pi bond of stannaacetylene ; the structures of compounds and were fully characterized by X-ray crystallography.

A dendrimer approach to high-spin polycarbenes. Conversion of connectivity from disjoint to non-disjoint by perturbation of nonbonding molecular orbital coefficients.

[structure: see text] Pentakis(diazo) compound was prepared by coupling 3,5-bis[4-[diazo(4-tert-butyl-2,6-dimethylphenyl)methyl]-3,5-dibromophenylethynyl]phenylacetylene with bis(4-iodo-2,6-dimethylphenyl)diazomethane under Sonogashira reaction conditions. Pentakis(carbene) generated by irradiation of the pentakis(diazo) compound was shown to have a high-spin state with S = 4.4 at 2.0 K.

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes.

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.

Preparation of sterically congested di(4-pyridyl)diazomethanes and characterization of triplet carbenes from them.

Di(4-pyridyl)diazomethanes having two and four ortho chlorine groups were prepared, and the triplet carbenes generated from them were characterized by ESR and UV/vis at low temperature and time-resolved UV/vis at room temperature. An appreciable increase in the stability of triplet carbenes is achieved by introducing ortho chlorine groups. [structure: see text]

Preparation of a copper ion complex of sterically congested diphenyldiazomethanes having a pyridine ligand and characterization of their photoproducts.

To show that persistent high-spin polycarbenes can be realized by utilizing hetero spin systems, two diphenyldiazomethanes having pyridyl groups, i.e., bis{4-(4-pyridyl)-2,6-dimethylphenyl}diazomethane (4,4'-DPy-1-N(2)) and {2,4-di(4-pyridyl)-6-bromophenyl}(2,6-dimethyl-4-tert-butylphenyl)diazomethane (2,4-DPy-1-N(2)), were prepared. Triplet carbenes, 4,4'-DPy-1 and 2,4-DPy-1, generated by photolysis of the corresponding diazomethanes were characterized by spectroscopic means (ESR and UV/vis in matrix at low temperatures and laser flash photolysis in solution at room temperature). The results showed that they were fairly persistent. Magnetic properties of the photoproducts from a 1:1 complex between DPy-1-N(2) and Cu(hfac)(2) (hfac = hexafluoroacetylacetonate) were characterized by ESR and a superconducting quantum interference device (SQUID) magneto/susceptometer. The field dependences of magnetization for the complexes, expressed by using M versus H/T plots, were analyzed in terms of the Brillouin function to be S = 6.80 (F = 0.60) for the 1:1 complex of 4,4'-DPy-1 and Cu(hfac)(2) and S = 3.71 (F = 0.73) for the 1:1 complex of 2,4-DPy-1 and Cu(hfac)(2) at 2.0 K. Thus, it has been demonstrated that a high-spin species is actually generated in the photoproducts and that the complexed carbenes showed significant stability.

Emission spectroscopic investigation of triplet diarylcarbene generated in molecular sieve VPI-5.

We report an attempt to generate and characterize a triplet carbene, bis(2,4,6-trichlorophenyl)carbene ((3)1), in zeolites Y and L and in a molecular sieve VPI-5 in which a possible dimerization and reaction with the precursor of carbene are significantly retarded, thus making the triplet carbene longer lived than in solution at room temperature. The adsorption of a corresponding diazomethane (1-N(2)), the precursor of 1, was carefully examined by comparing the absorption spectrum after adsorption with that of 1-N(2) in n-pentane, which revealed that 1-N(2) was adsorbed with the diazo group intact only in VPI-5, while in other zeolites 1-N(2) was found to be decomposed upon adsorption. This difference in reactivity of the hosts was ascribed to the absence of Brønsted-acid sites in VPI-5. The photoirradiation of 1-N(2) in VPI-5 at 77 K was monitored by emission spectroscopy, which revealed that bis(2,4,6-trichlorophenyl)methyl radical (1-H) was produced as the only detectable species under these conditions. This is interpreted as indicating that nascent (3)1 may undergo efficient hydrogen abstraction as a result of multiple excitation by repeated refraction and reflection of the light in a light-scattering medium. In accord with this interpretation, the emission due to (3)1 was observed when irradiation was carried out on a translucent glassy sample prepared by submerging VPI-5 incorporating 1-N(2) in a refractive-index-matching fluid such as propylene glycol or glycerol. ESR signals ascribable to (3)1 were also observed under these conditions. Laser photolysis of 1-N(2) in VPI-5 at room temperature with fast detection of both emission and absorption showed that the bands due to 1-H were detected in the nanosecond time regime probably because of the extremely fast H abstraction by (3)1. However, a variable-temperature ESR study showed that the signals due to (3)1 survive up to 220 K in VPI-5 while the signals disappear at 120 K in 2-methyltetrahydrofuran, suggesting that triplet carbene is stabilized in VPI-5. Thus, a triplet carbene was generated and characterized in a zeolite for the first time and shown to be stabilized extrinsically. The present study also proposes a solution to the issues of acidic sites and multiple excitation often observed in zeolites.

[Discrepancies between laparoscopic and histological findings in the stage diagnosis of chronic viral hepatitis].

We studied about the discrepancies of stage diagnosis between laparoscopic and histological findings in the clinical course of chronic viral hepatitis. We noticed discrepancies in 26% of chronic hepatitis B and 18% of chronic hepatitis C. Many cases were judged more advanced by laparoscopic staging than by histological staging. The group with different stage diagnosis showed high frequency of reddish markings and patchy markings indicating severe necro-inflammatory reaction and regenerative reaction in laparoscopic findings. This suggests that existence of active inflammation might be a cause of discrepancies in stage diagnosis. The cumulative incidence of hepatocellular carcinoma was significantly higher in the cases judged more advanced by laparoscopy even in the same histological stage. This indicates that laparoscopic staging should be more reliable for predicting prognosis in each patient.

Generation, direct observation, and kinetics of triplet 9-(1,1,4,4,5,5,8,8-octamethyl-1,2,3,4,5,6,7,8-octahydro- anthryl)phenylcarbene.

[structure: see text] Reactivities of the title triplet carbene were compared with those of the "untied" counterpart, [2,4,6-tris(tert)butylphenyl](phenyl)carbene. An appreciable increase in the stability of triplet carbene is noted by tuning the distance between the o-hydrogen and the carbene center.

Quintet bis(carbene) from triplet diarylcarbenes introduced at the 1,8-position of anthracene.

[9-(10-phenyl)anthryl](4-bromo-2,6-dimethylphenyl)diazomethane was found to be stable enough to survive Sonogashira coupling reaction conditions and was converted to [9-(10-phenyl)anthryl](4-trimethylsilylethynyl-2,6-dimethylphenyl)diazomethane, which was reacted with 1,8-diiodoanthracene to give bis(diazo) compound. Bis(carbene) generated by irradiation of the bis(diazo) compound generated a fairly persistent S = 2 quintet state. [structure: see text]

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death.

Loss of ABCB7 gene: pathogenesis of mitochondrial iron accumulation in erythroblasts in refractory anemia with ringed sideroblast with isodicentric (X)(q13).

An isodicentric (X)(q13) (idicXq13) is a rare, acquired chromosomal abnormality originated by deletion of the long arm from Xq13 (Xq13-qter), and is found in female patients with hematological disorders involving increased ringed sideroblasts (RSs), which are characterized by mitochondrial iron accumulation around the erythroblast nucleus. The cause of increased RSs in idicXq13 patients is not fully understood. Here, we report the case of a 66-year-old female presenting with refractory anemia with ringed sideroblasts (RARS), and idicXq13 on G-banded analysis. We identify the loss of the ABCB7 (ATP-binding cassette subfamily B member-7) gene, which is located on Xq13 and is involved in mitochondrial iron transport to the cytosol, by fluorescent in situ hybridization (FISH) analysis and the decreased expression level of ABCB7 mRNA in the patient's bone marrow cells. Further FISH analyses showed that the ABCB7 gene is lost only on the active X-chromosome, not on the inactive one. We suggest that loss of ABCB7 due to deletion of Xq13-qter at idicXq13 formation may have contributed to the increased RSs in this patient. These findings suggest that loss of the ABCB7 gene may be a pathogenetic factor underlying mitochondrial iron accumulation in RARS patients with idicXq13.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain.

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

Overexpression of a host factor TOM1 inhibits tomato mosaic virus propagation and suppression of RNA silencing.

A plant integral membrane protein TOM1 is involved in the multiplication of Tomato mosaic virus (ToMV). TOM1 interacts with ToMV replication proteins and has been suggested to tether the replication proteins to the membranes where the viral RNA synthesis takes place. We have previously demonstrated that inactivation of TOM1 results in reduced ToMV multiplication. In the present study, we show that overexpression of TOM1 in tobacco also inhibits ToMV propagation. TOM1 overexpression led to a decreased accumulation of the soluble form of the replication proteins and interfered with the ability of the replication protein to suppress RNA silencing. The reduced accumulation of the soluble replication proteins was also observed in a silencing suppressor-defective ToMV mutant. Based on these results, we propose that RNA silencing suppression is executed by the soluble form of the replication proteins and that efficient ToMV multiplication requires balanced accumulation of the soluble and membrane-bound replication proteins.