Electromyography of the cricoarytenoid unit during supracricoid laryngectomy with a cricohyoidoepiglottopexy procedure.

Two patients who received supracricoid laryngectomy with cricohyoidoepiglottopexy to treat laryngeal cancers, underwent intra-operative electromyography analysis. After the lesion was removed and the electrodes were inserted into the remaining intrinsic laryngeal muscles, the depth of anaesthesia was carefully reduced. Gentle tactile stimulations were applied to the pharynx to trigger the reflex movement of the remaining arytenoids. Recordings were made when reflex movement was achieved. Case one: Electromyography (EMG) of the remaining arytenoid demonstrated clear phase differences indicating reciprocal activities between the adductor group (lateral cricoarytenoid muscle, interarytenoid muscle) and the abductor muscle (posterior cricoarytenoid muscle). Case two: EMG of the remaining arytenoid demonstrated reciprocal activities between the interarytenoid muscle and the posterior cricoarytenoid muscle. Activity of the lateral cricoarytenoid muscle was not evident because the muscle was excised during removal of the paraglottic space. Mobility of the arytenoid was attributed to interaction between the interarytenoid muscle and posterior cricoarytenoid muscle. Reciprocal interaction between the interarytenoid muscle and posterior cricoarytenoid muscle alone is also capable of maintaining post-operative laryngeal functions after supracricoid laryngectomy with cricohyoidoepiglottopexy.

Sugar conformation of a stereospecific 2'-R or 2'-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants.

The sugar conformation of a DNA decamer was studied with proton-proton 3J coupling constants. Two samples, one comprising stereospecifically labeled 2'-R-2H for all residues and the other 2'-S-2H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980-6986; Nucleosides Nucleotides (1995) 14, 333-336], the deuterium labeling being highly stereospecific (> or = 99% for all 2''-2H, > or = 98% for 2'-2H of A, C, and T, and > or = 93% for 2'-2H of G). The 3J values of all H1'-H2' and H1'-H2'' pairs, and several H2'-H3' and H2''-H3' pairs were determined by line fitting of 1D spectra with 0.1-0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3'-exo and C2'-endo with phi(m) values of 26 degrees to 44 degrees, except for the second and 3' terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from JH1'H2' and JH1'H2'' values. For C10, the N-S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1'-exo conformation with 27 degrees distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms.

Different chemical methods used to attach oligonucleotides by their 5'-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5'-specific linkage that satisfyingly withstands PCR conditions and leaves the 3'-ends available for DNA polymerase activity. The best results were obtained with 5'-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5'-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.

Design, synthesis, and antiviral evaluation of 2-deoxy-D-ribosides of substituted benzimidazoles as potential agents for human cytomegalovirus infections.

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride was achieved to give the desired beta nucleosides 2b-h. Subsequent deprotection afforded the corresponding free beta-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-beta-D-erythro- pentofuranosyl)benzimidazole (10) was prepared using 2'-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.

Study on highly diastereoselective synthesis of (2'R)-2'-deoxy[2'-2H]guanosine.

To develop an efficient method for the synthesis of a highly diasteroselective (2'R)-2'-deoxy[2'-2H]guanosine (1), studies of organic chemical conversion from 2'-bromo-2'-deoxy-N2-Isobutyryl-3',5'-O-TIPDS-guanosine (2) to 1 and a biological transdeoxyribofuranosylation of (2'R > 98% de)-2'-deoxy[2'-2H]uridine (4) were carried out. As the results, a highly diastereoselective synthesis of 1 was achieved by a biological transdeoxyribofuranosylation between 2,6-diaminopurine and 4 by the use of Enterobacter aerogenes AJ-11125, followed by treatment with adenosine deaminase. The results will be described in detail.

Synthesis of oligonucleotides using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-H-phosphonate and -H-phosphonothioate derivatives.

Synthetic studies on phosphodiester, phosphorothioate, and phosphorodithioate-linked oligonucleotides in terms of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) group as the base-labile protecting group for the 5'-hydroxyl groups of nucleoside 3'-H-phosphonate and -H-phosphonothioate derivatives, are described.

Contribution of individual subunits to the multimeric P2X(2) receptor: estimates based on methanethiosulfonate block at T336C.

P2X receptors are membrane proteins that incorporate a cation-selective ion channel that can be opened by the binding of extracellular ATP. They associate as hetero- and homo-multimers of currently unknown stoichiometry. In this study, we have used Xenopus laevis oocytes to express rat P2X(2) receptor subunits, which carry a cysteine mutation at position 336. ATP-induced currents at this mutant receptor subunit were blocked by more than 90% when exposed to [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), whereas currents from wild-type subunits were not affected. To compare mutant and wild-type channel expression, we introduced an epitope in their extracellular domains and found for both channels a similar linear relationship between antibody binding and currents induced by ATP. To study the contribution of the individual subunits to the block by MTSET, we coinjected different mixtures of wild-type and mutant-encoding mRNAs. We found that the inhibition by MTSET depended linearly on the proportion of mutant subunits, which was clearly contrary to the hypothesis that a single mutant subunit could act in a dominant fashion. Subsequent concatenation of wild-type and mutant-encoding cDNAs resulted in an inhibition by MTSET that also depended linearly on the number of mutant subunits and was independent of the position of the mutant subunit, as long as only two or three P2X(2) subunits were joined. With four or six subunits joined, however, the inhibition by MTSET became strongly position-dependent. The present results show that a "per-subunit" channel block causes the blocking effects of MTSET and they suggest that not four but maximally three subunits actively participate in the channel formation.

[Efficient synthesis of nucleosides labeled with stable isotopes and their application to structural biology].

1) A series of synthetic works on nucleosides appropriately labeled with stable isotopes of deuterium, carbon-13, and nitrogen-15 has been undertaken, confronting the strong demands for the nucleosides in the NMR spectroscopic study deeply related to structural biology, and the synthetic methods of (2'R)- and (2'S)-2'-deoxy[2'-2H]ribonucleosides, 2'-deoxy[5'-2H]ribonucleosides, [5'-13C]ribonucleosides, 2'-deoxy[5'-13C]ribonucleosides, 2'-deoxy[4-15N]cytidine, [4-15N]cytidine, 2'-deoxy[6-15N]adenosine, and [6-15N]adenosine were developed more efficiently than ever; some oligodeoxyribonucleotides were constructed by the use of these materials and found to be extraordinarily feasible for the NMR spectroscopic studies. 2) A novel approach to oligonucleotide synthesis on CPG has been established by the use of a base-labile protecting group, i.e., 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting group for the 5'-hydroxyl group of nucleoside 3'-phosphoramidites.

Structural analysis of the complex of a distamycin analogue with the Dickerson dodecamer 13C labeled at 5'-carbons using NMR spectroscopy.

Structural analysis of the complex of a distamycin analogue (Tallimustine) with the Dickerson dodecamer d(C*G*C*G*A*A*T*T*C*G*C*G) [N*:[5'-(13)C]nucleotide] was performed by NMR spectroscopy and the results will be described in detail.

[Left atrial function and left atrial appendage flow velocity in hypertrophic cardiomyopathy: comparison of patients with and without paroxysmal atrial fibrillation].

The involvement of left atrial (LA) appendage flow velocity in reduced left atrial function was investigated in 24 patients with hypertrophic cardiomyopathy, who retained sinus rhythm at the examination. Patients were divided into 11 with a history of paroxysmal atrial fibrillation [PAf(+)] and 13 without such history [PAf(-)]. Transthoracic echocardiography was performed to evaluate LA fractional shortening (LA%FS) and mean velocity of circumferential LA fiber shortening (LAmVcf), as contractile functions of the left atrium at the phase of active atrial contraction. Transesophageal echocardiographic Doppler examination was performed in all patients to measure the LA appendage velocity. In all patients, significant positive correlations were observed between the LA appendage velocity and LA%FS (r = 0.50, p < 0.05) or LAmVcf (r = 0.82, p < 0.001). LAmVcf and LA appendage velocity in patients with paroxysmal fibrillation were significantly lower than in those without (0.84 +/- 0.15 vs 1.28 +/- 0.37 circ/sec, 44 +/- 12 vs 65 +/- 20 cm/sec, both p < 0.01), whereas LA diameter was greater in the former compared to the latter (45 +/- 5 vs 38 +/- 5 mm, p < 0.01). LAmVcf and LA appendage velocity were low in four patients with cerebral infarction or transient cerebral ischemic attack (LAmVcf < 1.0 circ/sec, LA appendage velocity < or = 40 cm/sec). Importantly, all these patients had a history of paroxysmal fibrillation. These results indicate that there is a close relationship between LA appendage velocity and LA contractile function in patients with hypertrophic cardiomyopathy with paroxysmal atrial fibrilation, and these patients have potential risk of cerebral infarction.

A case of cardiac sarcoidosis: significance of ventricular tachycardia originating from the septum.

A 65-year-old woman was admitted for assessment of recurrent tachycardia. Cross-sectional echocardiography showed that the anterobasal portion of the ventricular septum was thin and dyskinetic. An electrophysiologic study revealed ventricular tachycardia, during which marked fragmented potentials could be obtained from the anterior septal aspect of the right ventricle. The site of earliest activation was in the vicinity of the His bundle. A diagnosis of cardiac sarcoidosis was made by based on endomyocardial biopsy combined with the clinical manifestations. Ventricular tachycardia originating from the anterior septum may be an indicator of underlying cardiac sarcoidosis.

A novel and efficient method for the stable expression of heteromeric ion channels in mammalian cells.

The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.

[Significance of coronary risk factors and coronary arteriosclerosis for coronary vasospasm].

The clinical significance of coronary arteriosclerosis and coronary risk factors was investigated in patients with coronary spasm. Coronary spasm induction test with acetylcholine was performed in 140 consecutive patients (85 males and 55 females) with chest pain in our hospital. The patients were divided into positive, borderline, and negative groups according to the results of the test. The positive and borderline groups were categorized as the coronary contractive group. The coronary sclerosis index was used to evaluate the degree of coronary arteriosclerosis. Coronary risk factors were evaluated in terms of hypertension, diabetes mellitus, hyperlipidemia, obesity, history of smoking and drinking, and family history of cardiovascular events. Patients could be divided into 34.3% in the positive group and 23.6% in the borderline group, i.e. 57.9% in the coronary contractive group, and 42.1% in the negative group. There were more males than females in both positive and coronary contractive groups. The proportion of males in the coronary contractive group was higher in patients over 60 years of age than in patients under 60. In contrast, the proportion of females was higher in patients under 60 than in patients over 60. In male patients, the coronary sclerosis indices in the positive, borderline, and coronary contractive groups were higher than those in the negative group. The indices in female patients in the positive and coronary contractive groups were higher than the index in the negative group. There were no differences in terms of the presence or absence, or the degree of organic stenosis between spastic sites and nonspastic branches in the positive group. The history of smoking in male patients was significantly more common in the positive group than in the negative group. The family history was more relevant in female patients in the positive or coronary contractive group compared to the negative group. Moreover, the history of smoking in the coronary contractive group was significantly more common than that in the negative group. The development of coronary spasm may be determined, at least in part, by the degree of coronary sclerosis as well as by gender and age. Smoking habits in both sexes and family history in females are proposed as the most important risk factors for coronary spasm.

Isolated right ventricular hypoplasia: intraoperative balloon occlusion test.

A 42-year-old woman with isolated right ventricular hypoplasia underwent operation. To determine the optimal procedure, we temporarily occluded the atrial septal defect with a balloon catheter that was introduced through the right atrial appendage. The hemodynamics were stable throughout the occlusion test. Simple closure of the atrial septal defect was safely accomplished. Intraoperative occlusion test with the balloon catheter was a good alternative to preoperative occlusion test.

Stereospecific assignment of H5' and H5″ in a (5'R)-/(5'S)-deuterium- labeled DNA decamer for(3) J (HH) determination and unambiguous NOE assignments.

The stereoselective deuterium labeling at the 5' methylene protons of the ribose ring recently developed by Kawashima et al. [1995, Tetrahedron Lett., 36, 6699-6700] enabled the assignment of pro-R and pro-S protons at the 5' position. The deuterium-labeled nucleotides, [(5'S)-(2)H]- and [(5'R)-(2)H]-diastereomers, in an approximate ratio of 2:1, were incorporated in the decamer 5'-d(GCATTAATGC)-3'. Thus, both pro-R and pro-S methylene proton signals without geminal coupling appeared in the NOESY and DQF-COSY spectra. Complete stereospecific assignments and simplified spin systems enabled the determination of 15 (3)J coupling constants between H4' and H5'/H5", and the unambiguous assignment of 135 NOESY cross peaks originating from H4'/H5'/H5" resonances.

Tissue distribution of the P2X7 receptor.

The P2X7 receptor is a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules as large as propidium dyes (629 Da). In situ hybridization using a digoxigenin-labelled riboprobe, and immunohistochemistry using an antibody raised against a C-terminal peptide sequence, were used to determine the distribution of the P2X7 receptor mRNA and protein in rat and mouse tissues and cell lines. The brain of newborn rats showed a 6 kb RNA by Northern blotting, but this was not detectable in adult brain. By in situ hybridization and immunohistochemistry, there was heavy labelling of ependymal cells in both newborn and adult brain, but the brain parenchyma showed no labelling. However, P2X7 receptor-immunoreactive cells appeared in the penumbral region around an area of necrosis evoked by prior occlusion of the middle cerebral artery, suggesting expression of the receptor by activated microglia. NTW8 cells, a mouse microglial cell line, strongly expressed the P2X7 receptor mRNA and protein. The P2X7 receptor mRNA and protein were also observed in the majority of bone marrow cells, including those separately identified by their expression of other antigens as granulocytes, monocyte/macrophages and B lymphocytes. The expression of P2X7 receptor by brain macrophages rather than neurons would be consistent with a role in brain repair following inflammation, infarction or immune insult.

Baculovirus expression provides direct evidence for heteromeric assembly of P2X2 and P2X3 receptors.

P2X2 and P2X3 are subunits of P2X receptors, cation channels opened by binding extracellular ATP. cDNAs encoding P2X2 and P2X3 receptor subunits, each with one of two C-terminal epitope tags, were cloned into baculovirus. Virally infected insect cells (Spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by Western blotting, by the specific binding of [35S]ATP and by whole-cell recordings of membrane current evoked by ATP or alphabetamethylene-ATP. In cells infected at the same time with two viruses encoding P2X2 and P2X3 receptors, the two proteins could be cross-immunoprecipitated with antibodies specific for either of the epitope tags. Whole-cell recordings from these cells showed that ATP and alphabetamethylene-ATP evoked currents with agonist sensitivity and desensitization quite distinct from those observed when P2X2 or P2X3 receptors were expressed alone. The results offer a method to express large amounts of P2X receptor protein, and they provide direct evidence that P2X2 and P2X3 subunits assemble to form heteromeric channels having distinct properties from those formed as homomers.

[Secretion of atrial natriuretic peptide from the left ventricle and heart in patients with hypertrophic cardiomyopathy: relationship with hemodynamic and echocardiographic profiles].

This study investigated whether atrial natriuretic peptide (ANP) secretion from the left ventricle occurs and correlated any secretion with the hemodynamic or echocardiographic parameters in patients with hypertrophic non-obstructive cardiomyopathy (HNCM). Volume overload was induced by intravenous injection of low-molecular weight dextran in 19 patients with HNCM and seven normal subjects (control group). Simultaneous measurements of plasma ANP concentrations in the aortic root, anterior interventricular vein and coronary sinus, and hemodynamic parameters were performed before and after volume overload. Echocardiographic parameters were measured under the basal conditions in the HNCM group. There was no difference in ANP concentrations between the aorta and anterior interventricular vein either before or after volume overload in the control group, whereas they were significantly higher in the anterior interventricular vein than in the aorta both before and after volume overload in the HNCM group, suggesting increased ANP secretion from the left ventricle. Moreover, the difference in ANP concentrations between these locations was enhanced by volume overload. In the HNCM group, the differences in ANP concentrations between these locations were positively correlated with the pulmonary capillary wedge pressure and left ventricular end-diastolic pressure before and after volume overload, but not with left ventricular thickness or left atrial dimension. These results suggest that enhanced ANP secretion from the left ventricle in both basal and volume overloaded states occurs in HNCM, and this ANP secretion from the left ventricle is closely related to left ventricular end-diastolic pressure but not to wall thickness and mass index of left ventricle.

Highly diastereoselective synthesis of 2'-deoxy[2'-2H]ribonucleoside derivatives by the use of tris(trimethylsilyl)[2H]silane.

The use of (Me3Si)3SiH or (Me3Si)3Si2H-Et3B system in place of Bu3SnH or Bu3Sn2H-Et3B system for a reductive protylation or deuteration reaction of 2'-Br-2'-deoxy-3',5'-O-TIPDS-[2'-2H]ribonucleosides or 2'-Br-2'-deoxy-3',5'-O-TIPDS-ribonucleosides was confirmed to improve the reactions to afford an excellent diastereoselectivity, i.e., the diastereoselectivity of (2'S)- or (2'R)-2'-deoxy-[2'-2H]ribonucleosides obtained by the reaction at 0 degree C is equivalent to the diastereoselectivity obtained by that using Bu3SnH or Bu3Sn2H-Et3B system at < -70 degrees C; the reductive deuteration of 2'-O-phenoxythiocarbonyl-3',5'-O-TIPDS-ribonucleosides through (Me3Si)3Si2H-Et3B system at 0 degree C also gave (2'R)-2'-deoxy[2'-2H]ribonucleosides with such a high diastereo-selectivity as above.

[Partial rupture progressing to complete rupture of the left ventricular anterior papillary muscle after acute myocardial infarction: a case report].

A 71-year old man presented with partial rupture progressing to complete rupture of the left ventricular anterior papillary muscle after acute anterolateral myocardial infarction. The progressive rupture was demonstrated by transthoracic and transesophageal echocardiography. Transthoracic echocardiography showed exaggerated systolic prolapse of the anterior mitral leaflet with grade III mitral regurgitation and partial disruption of the anterolateral papillary muscle, but transesophageal echocardiography during surgery disclosed the progression of the partial rupture to complete rupture. The flail anterior mitral leaflet with severe mitral regurgitation and the head of the ruptured papillary muscle into the left atrium in systole were confirmed. The patient was treated by coronary artery bypass grafting and mitral valve prosthesis using a St. Jude Medical valve with good outcome.