[Endarterectomy and aortic valve replacement in a patient with severely calcified ascending aortic root].

A severely calcified, so-called, "porcelain" aorta may complicate aortic valve replacement (AVR). We report a case of successful AVR with an extensively calcified aortic root, involving the right coronary ostium. A 73-year-old female presented with a stenotic aortic valve with the pressure gradient of 60 mmHg. Computed tomography demonstrated extensive calcification of the ascending aortic root. Coronary angiogram showed 90% right coronary artery ostial stenosis. We endarterectomized the ascending aorta, the Valsalva sinus and the right coronary ostium, which enabled AVR with a 19 mm CarboMedics valve. These procedures might be a favorable option for the treatment of porcelain aorta and calcified aortic valve.

Gallbladder adenoma: report of a case with emphasis on contrast-enhanced US findings.

There is a marked paucity of contrast-enhanced ultrasound (US) findings of gallbladder disease in the literature, and there is only one previous case of gallbladder adenoma. We report such a case. US showed a 2-cm polypoid lesion at the gallbladder body. Color Doppler US showed the hypervascular nature of the lesion, and contrast-enhanced US revealed the lesion to be homogeneously enhanced, suggesting that the lesion was composed of the same pathology. The lesion was surgically resected, and was found to be an adenoma without cancer foci. This case suggests that contrast-enhanced US is an effective tool in diagnosing a gallbladder adenoma.

Duodenal adenoma with production of massive mucus: report of a case with emphasis on US findings.

Duodenal adenoma with massive mucus production is very rare. We report such a case. Ultrasonography (US) showed the presence of massive mucus, and contrast- enhanced US revealed the thickened wall to be homogeneously enhanced, suggesting that the lesion was composed of the same pathology. The US results were confirmed histologically by endoscopically guided biopsy. Thus, contrast-enhanced US helps determine the biopsy point and determine good diagnostic strategies.

Characterization of allergen-specific monocyte-derived dendritic cells generated from monocytes by a single-step procedure: effect on naïve and memory T cells.

BACKGROUND: Dendritic cells are one of the most potent antigen-presenting cells and when pulsed with allergen can modulate allergen-specific T-cell responses. We sought to establish a single-step method by which to generate allergen-specific monocyte-derived dendritic cells (MoDCs). METHODS: Dermatophagoides farinae (Df)-prepulsed MoDCs were generated from monocytes by culturing with Df in the presence interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha simultaneously. Df-prepulsed MoDC were incubated with autologous naive and memory T cells in the absence of recall antigen, then proliferation and cytokine production by T cells was determined. RESULTS: Generation of allergen-prepulsed MoDCs was confirmed by examining expression of surface molecules. Df-prepulsed MoDC selectively induced proliferation of Df-specific T cells in the absence of recall antigen. Under these conditions, Df-prepulsed MoDCs augmented but did not alter the cytokine production profile. In addition, Df-prepulsed MoDCs activated naive T cells leading to proliferation and selective production of IFN-gamma in allergic patients but not in healthy subjects. CONCLUSIONS: These results suggest that Df-prepulsed MoDC generated from monocytes by a simple single-step manipulation can induce Df-specific cellular responses from both naive and memory T cells in the absence of recall antigen, and these cells potentially can be utilized as immune adjuvants in allergen-specific immunotherapy.

[Valvular heart surgery in osteogenesis imperfecta].

Osteogenesis imperfecta is a disease in which fragile bones readily cause fracture. Valvular disease concurrently develops. However, the surgery-related mortality rate is approximately 30%. In this study, we report 2 patients with osteogenesis imperfecta who underwent valvular heart surgery. Patient 1 was a 31-year-old male. He had previously been diagnosed as having osteogenesis imperfecta. Echocardiography suggested aortic valve insufficiency, and aortic valve replacement was performed. Patient 2 was a 59-year-old male. During admission, osteogenesis imperfecta was diagnosed. Echocardiography suggested mitral valve insufficiency, and mitral valve plasty was performed. In the 2 patients, intraoperative hemorrhage was marked. However, there were no fatal complications. We also reviewed the literature.

Aortic valve repair of congenital bicuspid aortic valve associated with aneurysm of the ascending aorta.

Two unusual cases of congenital bicuspid aortic valve associated with aneurysm of the ascending aorta are reported. One patient with a 7-cm ascending aortic dilatation and aortic regurgitation (AR) (II/IV), and another with a 6-cm ascending aorta and AR (III/IV), presented for treatment. Replacement of the ascending aorta and aortic valve repair were performed in both cases. Aortic valve repair included resection of the raphe, leaflet plication and subcommissural annuloplasty. Both patients had satisfactory results in the early postoperative period. Despite the promising outcomes after surgery in these patients, long-term changes in valve function and durability remain unknown. Additional close observation and monitoring are required before the procedure can be recommended as the standard of care.

A simulated molecular evolution from minimal catalytic domain of a group I ribozyme.

Catalysis of Group I intron ribozymes is carried out by its core region consisting of two helical domains P4-P6 and P3-P7. Recently, our laboratory showed that a mutant Group I ribozyme lacking both the P4-P6 domain and the base-triples can perform the trans-esterification reactions. The result demonstrates that the elements required for splicing are concentrated in the P3-P7 domain. Based on this result, we carried out in vitro selection experiment starting from newly constructed libraries in the ribozyme lacking the P4-P6 domain and the base-triples. This selection experiment showed unexpected divergency on the limited sequence space for the active ribozymes.

[A case of aconitine poisoning saved with cardiopulmonary bypass].

"Torikabuto" is a kind of plant which contains deadly poison. Its ingredient is aconitine alkaloids. We report a case of aconitine poisoning with fatal arrhythmia and acute pulmonary edema who was saved with cardio pulmonary bypass. A 41-year-old male ate to mistake "Torikabuto" for wild plant. He developed symptoms of dysarthria and admitted to our hospital. He developed ventricular tachycardia and fibrillation soon after his admission. Then he developed cardiogenic shock. He was resuscitated and supported with a percutaneous cardio pulmonary bypass. Ventricular tachycardia disappeared 24 hours after admittion. About 1 week later, cardio pulmonary bypass was terminated and about 3 months later, he discharged from our hospital.

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase.

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the Ki value of each enantiomer toward electric eel acetylcholinesterase. R-EPN (Ki = 6 microM) inhibited acetylcholinesterase much more effectively than S-EPN (Ki = 52 microM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The Km value for R-EPN decreased from 0.24 to 0.03 mM, and the Vmax value increased from 0.25 to 0.60 U/mg of protein. Vmax/Km values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions.

We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of approximately 100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 A (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleo-tides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 A. Comparison between the fluorescent properties of a pyrene-perylene pair with those of a widely used fluorescein-rhodamine pair showed that the pyrene-perylene FRET is suitable for hybridization assays.

Single-step single-molecule PCR of DNA with a homo-priming sequence using a single primer and hot-startable DNA polymerase.

We have previously reported that a protein library can be constructed by directly combining PCR amplification of a single DNA molecule and cell-free protein synthesis. To specifically amplify single DNA molecules, however, two-step PCR with nested primers was used. Here we describe a simpler method for single-step amplification of a single molecule. The method involves the use of both hot-startable DNA polymerase and a DNA template that has homo-priming sequences at both ends for amplification using a single primer. These two modifications greatly decreased the possibility of formation and subsequent accumulation, respectively, of primer-dimers that inhibit the amplification of target template. In addition, a high-fidelity DNA polymerase was successfully used, resulting in the significant reduction of the accumulation of mutations during amplification.

[Aortic valve papillary fibroelastoma in a patient with mitral valve regurgitation].

Papillary fibroelastoma is a rare cardiac tumour. We describe a patient with mitral valve regurgitation and aortic valve papillary fibroelastoma. The patient was 62-year-old woman. She was referred to us for surgical treatment of mitral valve. Preoperative echocardiography showed rheumatic mitral valve regurgitation (Sellers grade III) and it also demonstrated mobile masses of the aortic valve. At operation, mitral valve was repaired by a posterior annuloplasty. Through the aortotomy, small tumors were found to be attached to each cusps of the aortic valve and they were successfully removed. The histopathologic diagnosis was papillary fibroelastoma of the aortic valve. The postoperative course was uneventful.

New technique of posterior mitral annuloplasty.

We have developed a new technique of mitral annuloplasty applicable in patients with rheumatic mitral regurgitation. This technique is designed to advance anteriorly the posterior annulus and leaflet and to gain more coaptation area. Technical details of this procedure are presented.

In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation.

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein of a jellyfish Aequorea victoria was extensively diluted to one molecule per well, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequences obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and Escherichia coli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.

Recent structural studies of peptides in Japan.

Recent x-ray structure analyses of peptides in Japan were reviewed. A series of peptides containing an aminoisobutyric acid (Aib) residue was analyzed and their helical types were investigated. Two convenient methods to determine helical types were obtained. One method was obtained by calculating averaged helical parameters and the other was obtained by investigating meridional intensity distributions. The conformations of some peptides, such as aureobasidin E, ascidiacyclamide, synthetic analogues of heat-stable enterotoxin, were studied by an x-ray diffraction method, and in some cases, with the help of nmr spectroscopy and molecular mechanics calculations. Since these peptides have physiologically important activities, the structure-activity relationships of these peptides were discussed. Several other peptide structures and the polymorphism of amino acids are also reported.

Synthesis and structure-activity relationships of peptidyl alpha-keto heterocycles as novel inhibitors of prolyl endopeptidase.

The preparation and in vitro prolyl endopeptidase (PEP) inhibitory activity of a series of alpha-keto heterocyclic compounds is described. The design is based on the introduction of alpha-keto heterocycles at the C-terminal end of substrate-like peptides. Many of the compounds including those substituted with thiazole, benzothiazole, benzoxazole, imidazole, and pyridine groups exhibit IC50 potencies of PEP inhibition at nanomolar levels. Structure-activity studies of the C-terminal heterocyclic groups indicate the importance of an sp2 nitrogen atom at a beta-position from the adjoining ketone carbonyl group. This heterocyclic nitrogen atom would provide a critical hydrogen-bond interaction with the histidine residue of the catalytic triad in PEP. Our inhibitors would extend the generality of the alpha-keto heterocycle design to another serine protease.

A study on conformationally restricted sangivamycins and their inhibitory abilities of protein kinases.

Conformational restrictions of sangivamycin, a rather selective inhibitor of PKC, could be achieved by the use of the steric effect and the gauche effect of the substituents on the ribofuranose moiety. The conformational deviations obtained by these methods were found to nicely correlate with the inhibitory ability of PKC.

Alkylation reaction of phosphorus oxyacids.

Alkylation reactions of phosphorus oxyacids (i.e. phosphoric acid, phosphorous acid, hypophosphorous acid) were examined by means of several methods for the synthesis of the corresponding esters. These reactions were applied to the synthesis of oligonucleotide having phosphotriester bond or phosphonate diester bond.