Perylene-Cy3 FRET System to Analyze Photoactive DNA Structures.

We report a new Förster resonance energy transfer (FRET) system for structural analyses of DNA duplexes using perylene and Cy3 as donor and acceptor, respectively, linked at the termini of a DNA duplex via D-threoninol. Experimentally obtained FRET efficiencies were in good agreement with theoretical values calculated based on canonical B-form DNA. Due to the relatively long Förster radius, this system can be used to analyze large DNA structures, and duplexes containing photo-reactive molecules can be analyzed since perylene can be excited with visible light. The system was used to analyze a DNA duplex containing stilbene, demonstrating that in the region of the stilbene cluster the duplex adopts a ladder-like structure rather than helical one. Upon photodimerization between stilbene residues, FRET efficiencies indicated the reaction does not disturb DNA duplex. This FRET system will be useful for analysis of photoreactions of nucleobases as well as a wide range of nucleic acid structures.

Visible-Light-Triggered Cross-Linking of DNA Duplexes by Reversible [2+2] Photocycloaddition of Styrylpyrene.

Reversible photo-cross-linking of a DNA duplex through the [2+2] photocycloaddition of styrylpyrene is reported. Styrylpyrene moieties on d-threoninol linkers were introduced into complementary positions on DNA strands. Irradiation of the styrylpyrene pair in the duplex with visible light at λ=455 nm induced a [2+2] photocycloaddition between styrylpyrenes that cross-linked the two strands of the duplex. Two diastereomers were formed after [2+2] photocycloaddition as a result of rotation of the styrylpyrene residues. Also, the cycloreversion reaction was induced by UV light at λ=340 nm, which reversibly yielded the uncross-linked strands.

Terminus-free siRNA prepared by photo-crosslinking activated via slicing by Ago2.

We report the development of photo-crosslinked siRNA strands modified at each terminus with p-cyanostilbene. The siRNA was nuclease resistant and retained RNAi activity. We further studied the activation mechanism of the covalently-crosslinked siRNA. Interestingly Dicer, which is known to generate siRNA with overhanging 3' ends from the precursor siRNA, did not cleave the crosslinked siRNA at all. Our results suggest that the activation of the crosslinked siRNAs required cleavage by Argonaute2.

Hetero-Selective DNA-Like Duplex Stabilized by Donor-Acceptor Interactions.

We report on the characterization of a novel hetero-selective DNA-like duplex of pyrene and anthraquinone pseudo base pairs. The pyrene/anthraquinone pairs showed excellent selectivity in hetero-recognition and even trimers were found to form a hetero-duplex. Pyrene and anthraquinone moieties were tethered on acyclic D-threoninol linkers and linked to adjacent residues by using standard phosphoramidite chemistry. When pyrene and anthraquinone were incorporated at pairing positions in complementary strands of natural DNA oligonucleotides, the duplex was stabilized significantly. Moreover, a pyrene hexamer and an anthraquinone hexamer formed a stable artificial hetero-duplex without the assistance of natural base pairs. The pyrene/anthraquinone pair was so stable that even trimers formed a hetero-duplex under conditions in which natural DNA strands of three residues do not.

Efficiency of [2 + 2] photodimerization of various stilbene derivatives within the DNA duplex scaffold.

A DNA duplex was used as a scaffold to evaluate the intrinsic reactivity of [2 + 2] photodimerization between stilbene derivatives; the duplex pre-organizes the substrates avoiding the need for an association step. Unmodified stilbenes were first introduced at base-pairing positions on complementary DNA strands. The duplex was then irradiated with 340 nm UV light. HPLC analyses revealed that [2 + 2] photodimerization proceeded rapidly without side reactions. Thus, it was confirmed that the DNA duplex could be used as an ideal scaffold for [2 + 2] photodimerization of stilbenes. Next, we examined homo-photodimerization abilities of various stilbene derivatives. Homo-photodimerization of p-cyanostilbene, p-methylstilbazolium, and p-stilbazole occurred efficiently, whereas homo-photodimerization of p-dimethylaminostilbene and p-nitrostilbene did not proceed at all, probably because the reaction was quenched by dimethylamino and nitro groups. Time-dependent density functional theory calculations revealed that excitation energy was correlated with quantum yield. We further investigated hetero-photodimerization. These reactions were made possible by the use of two complementary oligodeoxyribonucleotides tethering different stilbene derivatives. Reactivities in hetero-photodimerization were highly dependent on the combination of derivatives. A high correlation was observed between the quantum yields and energy gaps of HOMO and LUMO between reactive derivatives. Unexpectedly, nitrostilbene, which was non-reactive in homo-photodimerization, cross-reacted with p-methylstilbazolium and p-stilbazole, both of which had close HOMO or LUMO with nitrostilbene. Evaluation of the intrinsic reactivity of homo- and hetero-photodimerization of stilbene derivatives was made possible by the use of DNA as a scaffold.

p-Stilbazole moieties as artificial base pairs for photo-cross-linking of DNA duplex.

In this study, we report a photo-cross-linking reaction between p-stilbazole moieties. p-Stilbazoles were introduced into base-paring positions of complementary DNA strands. The [2 + 2] photocycloaddition reaction occurred rapidly upon light irradiation at 340 nm. Consequently, duplex was cross-linked and highly stabilized after 3 min irradiation. The CD spectrum of the cross-linked duplex indicated that the B-form double-helical structure was not severely distorted. NMR analysis revealed only one conformation of the duplex prior to UV irradiation, whereas two diastereomers were detected after the photo-cross-linking reaction. Before UV irradiation, p-stilbazole can adopt two different stacking modes because of rotation around the single bond between the phenyl and vinyl groups; these conformations cannot be discriminated on the NMR time scale due to rapid interconversion. However, photo-cross-linking fixed the conformation and enabled discrimination both by NMR and HPLC. The artificial base pair of p-methylstilbazolium showed almost the same reactivity as p-stilbazole, indicating that positive charge does not affect the reactivity. When a natural nucleobase was present in the complementary strand opposite p-stilbazole, the duplex was significantly destabilized relative to the duplex with paired p-stilbazole moieties and no photoreaction occurred between p-stilbazole and the nucleobase. The p-stilbazole pair has potential as a "third base pair" for nanomaterials due to its high stability and superb orthogonality.

Assessment of a novel stentless mitral valve using a pulsatile mitral valve simulator.

BACKGROUND AND AIM OF THE STUDY: The study aim was to develop a novel stentless mitral valve (SMV) and to evaluate its performance, using an original pulsatile simulator developed specifically to analyze the hydrodynamic function of the mitral valve. METHODS: The SMV developed at the authors' institution consists of two major components: a large anterior leaflet with commissures, and a small posterior leaflet. The valve is formed by suturing the leaflets (made from bovine pericardium) to a flexible (Duran) ring. The SMV, constructed with a 27 mm flexible ring, was installed into the mitral valve simulator, after which the four papillary flaps of the two leaflets were sutured to artificial papillary muscles. The artificial ventricle was driven pneumatically at a pulse rate of 70 beats/min, with a systolic fraction of 35%. The mean flow, aortic pressure, and atrial pressure were adjusted to 4.5 1/min, 120/80 mmHg, and 10 mmHg, respectively. A 27 mm mechanical valve (MEV; St. Jude Medical Inc.) was employed as a control. The hydrodynamic performance of the SMV and MEV were investigated and compared. An echo-Doppler study was also performed. RESULTS: The waveforms of the SMV and MEV showed a similar pattern. The mean transvalvular flow was 4.7 +/- 0.4 1/min for the SMV, and 3.55 +/- 0.13 1/min for the MEV (p < 0.001). Mitral regurgitation was 5.07 +/- 1.15 and 3.78 +/- 0.35 ml/beat, respectively (p < 0.05). Echocardiographic data indicated that the regurgitant jet towards the left atrial model was none or trivial for the SMV, and trivial for the MEV. CONCLUSION: Within the environment of the mitral valve simulator, the novel SMV prepared from bovine pericardium demonstrated excellent performance characteristics, and may represent a potential future alternative for bioprosthetic stented mitral valves.

Chronic total occlusion of the left main coronary artery with normal left ventricular motion: the occluded site confirmed by three-dimensional computed tomography.

Total occlusion of the left main trunk (LMT) frequently results in sudden cardiac death. As a result, it is rarely observed on coronary arteriogram. There are only a few reports on chronic total occlusion of the LMT. Most patients present with recent, severe angina, but it is not easy to distinguish chronic total occlusion of the LMT from other types of severe coronary heart diseases. Here, we report a very rare case of chronic total occlusion of the LMT. The patient is a 38-year-old female with a history of three normal deliveries. Chronic total occlusion of the LMT was suspected on coronary arteriogram 2 years previously in the other hospital; however, she continued working as a part-time employee at a supermarket. She was referred to our hospital because of slightly increased effort angina and shortness of breath. The final diagnosis and the site of occlusion were determined by three-dimensional computed tomography (3-D CT). The patient underwent coronary artery bypass graft (CABG) surgery, and ischemic symptoms completely disappeared.

Late thrombosis of a drug-eluting stent overlapped in the in-stent restenotic lesion of a bare metal stent.

Late stent thrombosis occurred in the lesion of a sirolimus-eluting stent implanted 6 months previously for an in-stent restenosis lesion in the distal right coronary artery. Seventeen days before admission due to acute myocardial infarction this time, aspirin was discontinued for colon polypectomy. Ticlopidine had been discontinued 3 months before the discontinuation of aspirin. In drug-eluting stent era, the interventional strategy and antiplatelet therapy require long term attention.

Oozing type of left ventricular rupture via subepicardial aneurysm diagnosed 5 months after myocardial infarction.

A 71-year-old man visited our hospital complaining of increasing fatigue and exertional dyspnea. He had had severe epigastric pain for the past 5 months. On admission, chest radiogram showed marked cardiac dilatation and echocardiogram massive pericardial effusion with a small subepicardial aneurysm at the posterior wall of the left ventricle. An urgent pericardiocentesis removed 1300 ml of bloody effusion. The red blood cell count of the pericardial effusion was similar to that of the peripheral blood, and there were no abnormal findings on cytologic and bacteriological examinations. Coronary angiography showed a blunt occlusion of the mid-portion of the circumflex artery. Left ventricular angiogram revealed aneurysmal deformity of the left ventricular posterior wall. These findings suggested that an oozing type of left ventricular rupture via a subepicardial aneurysm had occurred after the onset of myocardial infarction (MI), resulting in massive accumulation of pericardial effusion. The patient is presently doing well without any clinical symptoms 18 months after pericardiocentesis. This is the first case report in which a subepicardial aneurysm with massive pericardial effusion was detected in the chronic stage of MI and successfully managed without surgical repair.

Systolic anterior motion (SAM) of the posterior mitral leaflet: left ventricular outflow tract obstruction in a patient without left ventricular hypertrophy.

Systolic anterior motion (SAM) of the anterior mitral leaflet with mitral-septal contact was generally thought to be a major contributor to dynamic left ventricular outflow tract obstruction in patients with hypertrophic cardiomyopathy. We report an interesting case of SAM of the posterior mitral leaflet in a patient without left ventricular hypertrophy, which led to dynamic left ventricular obstruction.