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.

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.

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.

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.

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.

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.

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.

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.

Structure determination of triplet diphenylcarbenes by in situ X-ray crystallographic analysis.

Crystalline-state photoreactions of the following diphenyldiazomethanes were investigated by in situ X-ray crystallography, spectroscopy, and theoretical calculations: bis(2,4,6-trichlorophenyl)diazomethane (1-N2), bis(2,4,6-tribromophenyl)diazomethane (2-N2), bis(2,6-dibromo-4-methylphenyl)diazomethane (3-N2), bis(2,6-dibromo-4-tert-butylphenyl)diazomethane (4-N2), (2,4,6-tribromophenyl)-(2,6-dimethyl-4-tert-butylphenyl)diazomethane (5-N2), bis(4-bromophenyl)diazomethane(6-N2), and diazofluorene (7-N2). Crystal structures of photoinduced triplet diphenylcarbenes (DPCs) of 1, 2, and 4 were determined. We found remarkable differences between their structural information obtained in the crystalline state and that previously obtained spectroscopically in a glass matrix. Although the triplet DPCs of 1, 2, and 4 have significantly different stabilities in solution, only subtle differences in their structural parameters, except for their C(:)-Ar bond lengths, are observed. It is noteworthy that the average bond length of C(:)-Ar for 4 (1.374 A) is considerably shorter than those for (3)1 and (3)2 (1.430 and 1.428 A, respectively), provided that the two C(:)-Ar bonds being compared were chemically equivalent. The most likely explanations for the small and large differences in bond lengths in 1, 2, and 4 may be derived from the packing effect. The packing patterns of 1 and 2 are identical, but that of 4 is totally different from those of 1 and 2. Moreover, these results are interpreted as indicating that triplet DPCs undergo relaxation upon softening of the environments. Theoretical calculations indicate that the potential energy surface of triplet DPCs in terms of the carbene angle is extremely flat and changes in the angles have little effect on the energies. Triplet DPCs with a sterically congested carbene center are trapped in a structure dictated by the precursor structure in a rigid matrix, even if this is not the thermodynamically most stable geometry, but undergo geometrical relaxation upon softening the matrix to relieve steric compression. ESR studies indicate that the interplanar angles are more flexible than the bond angles.

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.

Triplet diphenylcarbenes protected by trifluoromethyl and bromine groups. A triplet carbene surviving a day in solution at room temperature.

Two types of diphenyldiazomethanes having two trifluoromethyl and two bromine groups at the ortho positions, either in unsymmetrical or in symmetrical fashion, that is, (2,6-dibromo-4-phenylphenyl)[4-phenyl-2,6-bis(trifluoromethyl)phenyl]diazomethane (U-1-N2) and bis(2-bromo-4-phenyl-6-trifluoromethylphenyl)diazomethane (S-1-N2), are prepared. Triplet diphenylcarbenes (U-(3)1 or S-(3)1) are generated from those precursors and are characterized by ESR, UV/vis spectroscopy at low temperature, as well as time-resolved UV/vis spectroscopy at room temperature. Those carbenes are shown to be at least 2 orders of magnitude less reactive than the most stable triplet diphenylcarbene thus far known, that is, bis(2,6-dibromo-4-phenylphenyl)carbene. It has been also shown that S-(3)1 is significantly more stable than U-(3)1 even though both have the same two kinds of substituents. It is suspected that the perpendicular alignment of the two most bulky groups is a more effective way to shield the carbenic center than the planar one. By this way, triplet substituted diphenylcarbene surviving nearly a day in solution at room temperature is realized for the first time.

[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.

Preparation of sterically congested diphenyldiazomethanes having a pyridine ligand and magnetic characterization of photoproducts of their 2:1 copper complexes.

[structure: see text] To confirm whether high-spin species can be generated as a result of ferromagnetic interaction between the 3d spin of metal ions and the 2p spins of triplet carbene through the pyridyl group located remote from the carbene center, [2,6-dibromo-4-(3- and 4-pyridyl)phenyl](4-tert-butyl-2,6-dimethylphenyl)diazomethanes were prepared and the corresponding carbenes were generated either in the absence or presence of Cu(hfac)2. These were characterized by ESR and UV/vis in a matrix at low temperature, and by laser flash photolysis in solution at room temperature. These studies indicated that although both carbenes generated a fairly stable complex with copper ions, the 4-pyridyl isomer formed a high-spin species as a result of ferromagnetic interaction between the 3d spin of metal ions and the 2p spins of triplet carbene. Such an interaction in the corresponding 3-isomer is likely to be antiferromagnetic. This is further confirmed by magnetic measurements using a Superconducting Quantum Interference Device (SQUID). The results demonstrate that extension of this method will enable stable high-spin polycarbenes to be obtained.

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 copper ion complexes of sterically congested diaryldiazomethanes having a pyridine ligand and characterization of their photoproducts.

To realize fairly stable high-spin polycarbenes by utilizing heterospin systems comprising 2p spins of organic radicals and 3d spins of magnetic metal ions, we prepared dianthryldiazomethanes having two pyridyl groups at the 2,2'- or 2,7-positions, that is, bis[10-(4-tert-butyl-2,6-dimethylphenyl)-2-(4-pyridyl)-9-anthryl]diazomethane (2,2'-DPy-1-N(2)) and [10-(4-tert-butyl-2,6-dimethylphenyl)-9-anthryl][(10-(4-tert-butyl-2,6-dimethylphenyl)-2,7-di(4-pyridyl)-9-anthryl]diazomethane (2,7-DPy-1-N(2)). The triplet carbene DPy-(3)1 generated by photolysis of DPy-1-N(2) was characterized by ESR and UV-vis spectroscopy in a matrix at low temperature as well as by time-resolved UV-vis in solution at room temperature. The results showed that the triplet carbene DPy-(3)1 was destabilized to some extent as opposed to the parent triplet carbene before pyridination, but it was still fairly persistent, having a half-life of more than 30 min in solution at room temperature. Photoproducts from the complex between DPy-1-N(2) and Cu(hfac)(2) were characterized in a similar manner, and the results suggested that the generated carbene centers interacted magnetically with the Cu(II) ion to form a high-spin species with significant thermal stability. The fact that no significant signals due to the isolated triplet carbene DPy-(3)1 were observed suggested that the pyridine moiety binds with Cu(hfac)(2) in a nearly quantitative manner under these cryogenic conditions. Magnetic measurements of the photoproduct using a superconducting quantum interference device (SQUID) magneto/susceptometer were performed to determine the spin state of the complex. The temperature dependence of the molar paramagnetic susceptibility indicated the presence of ferromagnetic interaction. The field dependences of magnetization for the complexes, expressed using M versus H/T plots, were analyzed in terms of the two-component Brillouin function to be S = 3.18 (F = 0.66) and S = 0.02 (F = 0.23) for the 1:1 complex of 2,7-DPy-1 and Cu(hfac)(2) and S = 2.70 (F = 0.33) and S = 0.49 (F = 0.11) for the 1:1 complex of 2,2'-DPy-1 and Cu(hfac)(2).

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]