A metathesis reaction route to obtain fine Mg2Si particles.

We have developed a novel synthetic route for the production of fine Mg2Si particles (<1 µm) using NaSi, MgCl2, and Na. Mg2Si was suggested to be formed by a solid-state metathesis reaction, in which MgCl2 reacts with Na to form Mg and NaCl, and then Mg reacts with NaSi.

High-speed digital imaging laryngoscopy of the neoglottis following supracricoid laryngectomy with cricohyoidoepiglottopexy.

OBJECTIVES: This study aimed to analyse vocal performance and to investigate the nature of the neoglottal sound source in patients who had undergone supracricoid laryngectomy with cricohyoidoepiglottopexy, using a high-speed digital imaging system. METHODS: High-speed digital imaging analysis of neoglottal kinetics was performed in two patients who had undergone supracricoid laryngectomy with cricohyoidoepiglottopexy; laryngotopography, inverse filtering analysis and multiline kymography were also undertaken. RESULTS: In case one, laryngotopography demonstrated two vibrating areas: one matched with the primary (i.e. fundamental) frequency (75 Hz) and the other with the secondary frequency (150 Hz) at the neoglottis. In case two, laryngotopography showed two vibrating areas matched with the fundamental frequency (172 Hz) at the neoglottis. The interaction between the two areas was considered to be the sound source in both patients. The waveform of the estimated volume flow at the neoglottis, obtained by inverse filtering analysis, corresponded well to the neoglottal vibration patterns derived by multiline kymography. These findings indicated that the specific sites identified at the neoglottis by the present method were likely to be the sound source in each patient. CONCLUSIONS: High-speed digital imaging analysis is effective in locating the sites responsible for voice production in patients who have undergone supracricoid laryngectomy with cricohyoidoepiglottopexy. This is the first study to clearly identify the neoglottal sound source in such patients, using a high-speed digital imaging system.

In patients with ulcerative colitis, adsorptive depletion of granulocytes and monocytes impacts mucosal level of neutrophils and clinically is most effective in steroid naïve patients.

BACKGROUND: The aetiology of ulcerative colitis is inadequately understood, and drug therapy has been empirical rather than based on sound understanding of disease aetiology. This has been a major factor for refractoriness and adverse drug effects as additional complications. However, ulcerative colitis by its very nature is exacerbated and perpetuated by inflammatory cytokines, which are released by peripheral granulocytes and monocytes as well. Additionally, active ulcerative colitis is often associated with elevated peripheral granulocytes and monocytes with activation behaviour and are found in vast numbers within the colonic mucosa. Hence, from the clinicopathologic viewpoint, granulocytes and monocytes are appropriate targets for therapy in ulcerative colitis. Based on this thinking, an Adacolumn has been developed for depleting excess granulocytes and monocytes by adsorption. METHODS: By colonoscopy, biopsy and histology, we investigated the impact of granulocyte and monocyte adsorption (GMA) on the mucosal level of granulocytes and monocytes in patients with active ulcerative colitis. Forty-five patients (26 steroid naïve and 19 steroid-dependent), mean age 44.7 yr, were included. Twenty patients had total colitis and 25 had left-sided colitis. Each patient was given up to 11 GMA sessions over 12 weeks. No patient received additional medications within 4 weeks (steroid) to 8 weeks (other immunosuppressants) prior to entry or during the GMA course. Colonoscopy together with biopsy was done at entry and within 2 weeks after the last GMA session. RESULTS: At entry, the mean clinical activity index was 12.6; range 10-16. A total of 400 colonic biopsies were examined, which revealed massive infiltration of the colonic mucosa by granulocytes, and GMA was associated with striking reduction of granulocytes in the mucosa. At week 12, 33 of 45 patients (73.3%, P<0.01) had achieved clinical remission (the mean clinical activity index

Pitavastatin prevents bacterial translocation after nonpulsatile/low-pressure blood flow in early atherosclerotic rat: inhibition of small intestine inducible nitric oxide synthase.

BACKGROUND: Cardiopulmonary bypass decreases intestinal mucosal blood flow because of nonpulsatile and low-pressure blood flow resulting in bacterial translocation (BT) and atherosclerosis also has peripheral blood flow deficiency. The risk of nonpulsatile and low-pressure blood flow for atherosclerotic animals and the effect of statin administration, which has pleiotropic effects, were studied. METHODS: Wistar rats were divided into four groups: group N (normal diet), group C (high-cholesterol diet), group S (group C plus pitavastatin therapy), and group I [group C plus inducible nitric oxide (iNOS) inhibitor therapy]. First of all, vascular responses were measured. Then the rats underwent nonpulsatile/low-pressure blood flow in the intestine, and the serum peptidoglycan concentration as a parameter of BT, the small intestinal PO(2) ratio (intestinal PO(2)/PaO(2)) as a parameter of mucosal blood flow, and NO concentrations were measured before surgery (T0), at the end of 90 min of stenosis (T1), and 90 min after the release of stenosis (T2). Immunostaining for nitrotyrosine was also performed at T2. RESULTS: Group C had vascular endothelial dysfunction without histological changes, which indicated early atherosclerosis. The serum peptidoglycan concentration increased significantly at T2 only in group C. The intestinal PO(2) ratio was decreased at T1 in all the groups, and retuned to baseline at T2 in group N and group S, but not in group C or group I. Jejunal NO only in group C was significantly higher at all time points and ileal NO production at T1 and T2. There tended to be a positive stain for nitrotyrosine along the mucosal epithelium in group C. CONCLUSION: In the setting of early atherosclerosis, intestinal blood flow does not only improve after nonpulsatile/low-pressure blood flow but causes BT because of a large amount of NO from high enzymatic intestinal iNOS activity, and pitavastatin treatment can prevent BT by improving both issues.

Rapid and reagent-saving immunoassay using innovative stirring actions of magnetic beads in microreactors in the sequential injection mode.

We developed new ELISA techniques in sequential injection analysis (SIA) mode using microreactors with content of a few microliters. We immobilized antibodies on magnetic beads 1.0mum in diameter, injected the beads into microreactors and applied rotating magnetic fields of several hundred gauss. Magnetic beads, suspended in liquid in density of approximately 10(9)-10(10) particles per millilitre, form a large number of thin rod clusters, whose length-wise axes are oriented in parallel with the magnetic field. We rotate the Nd magnets below the center of the microreactor by a tiny motor at about 2000-5000rpm. These rotating clusters remarkably accelerate the binding rate of the antibodies with antigens in the liquid. The beads are trapped around the center of the rotating magnetic field even in the flowing liquid. This newly found phenomenon enables easy bead handling in microreactors. Modification of reactor walls with selected blocking reagents was essential, because protein-coated beads often stick to the wall surface and cannot move freely. Washing steps were also shortened.

[Assessment of extracellular fluid in cardiac surgery using cardiopulmonary bypass].

Extracellular fluid (ECF) was assessed before and after the cardiac surgery using cardiopulmonary bypass (CPB), by means of a bioimpedance spectrum analyzer to see volumes of the fluid based on changes of the impedance to various frequencies. Difference between the levels before and after the operation was divided by body weight to study about a % BW. Simultaneously its relation to the lung compliance [tidal volume/(peak inspiratory pressure-end expiratory pressure)] was studied. Mean age of the 18 patients was 59.1 +/- 19 years old. ECF was assessed before to 24 hours after the operation continuously and once more after 48 hours. Mean CPB time was 165 +/- 52 minutes, and aortic cross clamp time was 121 +/- 4 minutes. A remarkable increase of ECF was noted immediately after the operation, which further increased gradually till arriving at the peak 4 hours after the operation (4.52 +/- 1.8% BW). Then it gradually decreased to 0.641 +/- 2.7% BW 48 hours later. Lung compliance measured at the same time showed the lowest level 6 hours after the operation. It was known that the bioimpedance spectrum analysis is a simple and non-invasive method, which enables to monitor the vital stable before and after the operation.

Genomic RNA constellation of recently emerging serotype G14 equine rotavirus strains in Japan that is highly homologous with prototype G3 and G14 strains previously identified in the United States of America.

Serotype G14 was once considered to be uncommon among equine rotaviruses. While it sporadically emerged in some parts of the world, serotype G14 became the dominant G serotype among rotaviruses detected in foals with diarrhea in Japan in the late 1990s. However, it is not known how such recently emerging G14 rotaviruses are related in their overall genomic RNA constellation to prototype G14 strain identified earlier in the United States of America or how they were generated and why they have dominated over G3 equine rotaviruses. Genogrouping by RNA-RNA hybridization revealed that recently emerging serotype G14 equine rotavirus strains had an overall genomic RNA constellation that was highly conserved not only with contemporary and earlier G3 strains in Japan but also with prototype G3 and G14 strains previously identified in the United States of America. Japanese G14 rotavirus strains are likely to have originated form a VP7 gene substitution reassortant that had been formed earlier in the United States of America on the background of the then dominant G3 equine rotavirus.

Predominance of G3B and G14 equine group A rotaviruses of a single VP4 serotype in Japan.

A total of 65 equine group A rotaviruses (GAR) isolated from diarrheal foals at 48 farms in Hokkaido, Japan, between 1996 (29 isolates) and 1997 (36 isolates) were characterized for their VP7 and VP4 serotypes by PCR, nucleotide sequencing, and virus neutralization (VN) tests. By PCR VP7 typing, all isolates were classified as G3 or G 14, and the predominant serotype in each year was G3 (86%) in 1996 and G14 (53%) in 1997. VN tests with these 20 isolates randomly selected confirmed the specificity of PCR on the bases of complete agreement of the results in these methods (9 G3 and 11 G14), and revealed that all 9 G3 isolates were subtype G3B. There were five differing amino acid residues in three VP7 antigenic regions between subtypes G3A and G3B. Antiserum to a baculovirus recombinant that expressed P[12] VP4 neutralized all isolates and P[12] reference strains. These results suggest that genotype P[12] GAR belong to a single VP4 serotype, and that one VP4 and two VP7 serotypes (G3B and G14) of GAR were predominant in the equine population in Japan.

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses.

Detection of antibodies to the nonstructural protein (NS1) of influenza A virus allows distinction between vaccinated and infected horses.

Antibodies to the nonstructural protein (NS1) of A/equine/Miami/1/63 (H3N8) influenza virus were detected exclusively in the sera of mice experimentally infected with A/Aichi/2/68 (H3N2) and horses infected with A/equine/Kentucky/1/81 (H3N8) or A/equine/La Plata/1/93 (H3N8), but not in those of the animals immunized with the inactivated viruses, by enzyme-linked immunosorbent assay (ELISA) using a recombinant NS1 as antigen. The results indicate that the present method is useful for serological diagnosis to distinguish horses infected with equine H3 influenza viruses from those immunized with the inactivated vaccine.

Antigenic variation among equine H 3 N 8 influenza virus hemagglutinins.

To provide information on the antigenic variation of the hemagglutinins (HA) among equine H 3 influenza viruses, 26 strains isolated from horses in different areas in the world during the 1963-1996 period were analyzed using a panel of monoclonal antibodies recognizing at least 7 distinct epitopes on the H 3 HA molecule of the prototype strain A/equine/Miami/1/63 (H 3 N 8). The reactivity patterns of the virus strains with the panel indicate that antigenic drift of the HA has occurred with the year of isolation, but less extensively than that of human H 3 N 2 influenza virus isolates, and different antigenic variants co-circulate. To assess immunogenicity of the viruses, antisera from mice vaccinated with each of the 7 representative inactivated viruses were examined by neutralization and hemagglutination-inhibition tests. These results emphasize the importance of monitoring the antigenic drift in equine influenza virus strains and to introduce current isolates into vaccine. On the basis of the present results, equine influenza vaccine strain A/equine/Tokyo/2/71 (H 3 N 8) was replaced with A/equine/La Plata/1/93 (H 3 N 8) in 1996 in Japan. The present results of the antigenic analysis of the 26 strains supported the results of a phylogenetic analysis, that viruses belonging to each of the Eurasian and American equine influenza lineages have independently evolved. However, the current vaccine in Japan consists of two American H 3 N 8 strains; A/equine/Kentucky/1/81 and A/equine/La Plata/1/93. It is also therefore recommended that a representative Eurasian strain should be included as a replacement of A/equine/Kentucky/1/81.

Synthesis of cyclopentadienones catalyzed by methylidynetricobalt nonacarbonyl.

Easily prepared and air-stable methylidynetricobalt nonacarbonyl could be used as a catalyst for the intramolecular [2 + 2 + 1]-cocyclization of diynes and carbon monoxide producing cyclopentadienones.

Purification of horse immunoglobulin isotypes based on differential elution properties of isotypes from protein A and protein G columns.

Elution properties of horse immunoglobulin isotypes from protein A and protein G columns were examined. IgGa and IgGb isotypes were bound to protein A and protein G columns and were eluted by adjusting the pH of the elution buffer from 8.0 to 2.0. IgGc bound to protein G column but not to protein A column while IgG(T) bound to both columns. IgM and IgA apparently appeared not to bind to either column. New methods for purification of serum isotypes were developed using protein A and protein G columns as well as formerly established methods. Using these methods, it was possible to obtain purified isotypes for establishment of immunological assays for practical clinical use.

Pericardial hood repair of cardiac rupture secondary to extended myocardial infarction.

A surgical technique for simple and safe repair of oozing-type postinfarction cardiac rupture secondary to extended myocardial infarction is described. A hood-shaped pericardium was glued with gelatin-resorcinol and formaldehyde glue to cover the extended oozing infarcted myocardium. This technique was used on 3 elderly patients with good results.

The C-ring problem of sterol biosynthesis: TiCl(4)-induced rearrangement into the anti-Markovnikov cation corresponding to the C-ring.

[reaction: see text] Cation 9, generated by the reaction of diol 8 and BF(3).Et(2)O, SnCl(4), Sc(OTf)(3), FeCl(3), TiF(4), or CF(3)SO(3)H, leads to a hydride shift, providing cation 11, which corresponds to the initiation of backbone rearrangement. On the other hand, TiCl(4) selectively induces rearrangement to secondary cation 13 by ring expansion, which corresponds to the C-ring formation of sterol biosynthesis. AlCl(3) and ZrCl(4) induce further rearrangement into six-membered ring tert-cation 16.

In vitro detection of equine arteritis virus from seminal plasma for identification of carrier stallions.

Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU/ml. In vitro detection of EAV by virus isolation supplemented with RT-PCR using seminal plasma was proved to be an effective alternative to the standard test mating as a diagnostic method for carrier stallions.

Inhibitory effect of trehalose dimycolate (TDM) and its stereoisometric derivatives, trehalose dicorynomycolates (TDCMs), with low toxicity on lung metastasis of tumour cells in mice.

We examined the effect of semisynthetic trehalose-6,6 '-dimycolate (TDM) and its synthetic stereoisomeric derivatives (trehalose 6,6'-dicorynomycolates; TDCMs) prepared in oil-in-water (o/w) emulsion on inhibition of lung metastasis produced by highly metastatic murine tumour cells, colon 26-M3.1 carcinoma and B16-BL6 melanoma cells, using experimental and spontaneous metastasis models. Intravenous (i.v.) administration of TDM (100 microg/mouse) 1, 3 or 8 days before tumour inoculation significantly inhibited lung metastasis of colon 26-M3.1 cells, in a dose-dependent manner. Single administration of TDM 1 day after tumour inoculation also showed the therapeutic effect on experimental lung metastasis of colon 26-M3.1 cells. Similarly, multiple administrations of TDM after tumour inoculation resulted in a significant inhibition of spontaneous lung metastasis of B16-BL6 cells (on day 35), although it showed no effect on suppression of tumour growth (on day 21). In comparison of toxicity in vivo among TDM and four TDCMs such as TDCM(2R,3R), TDCM(2S,3R), TDCM(2R,3S) and TDCM(2S,3S), all of the TDCMs appeared to be less toxic than TDM itself. Furthermore, all of the TDCMs were prophylactically as well as therapeutically active for inhibition of lung metastasis of both colon 26-M3.1 and B16-BL6 tumour cells, showing higher inhibitory activity than that of TDM. In particular, TDCMs induced a marked suppression of the growth of B16-BL6 tumour cells in vivo. These results suggest that systemic administration of TDM as well as TDCMs led to inhibition of tumour metastasis and TDCMs are more potential to suppress tumour growth and inhibit tumour metastasis than TDM.

Analysis of the elephant trunk method in patients with extensive thoracic aortic aneurysm.

We assessed the appropriate length of an elephant trunk prosthesis based on our experience with 9 patients experiencing extensive thoracic aneurysms. There were 3 patients with a true aneurysm, 5 patients with a dissecting aortic aneurysm, and 1 patient with a true plus dissecting aortic aneurysm. The subjects were 4 men and 5 women and, at the time of operation, were from 38 to 74 years old. The second-stage operations were performed on 6 patients from 9 days to 6 months after the first-stage operation. In the first-stage operation, one patient died of pneumonia during the hospital stay and another died of multi-organ infarction after 15 months. In the second-stage operation, two patients died of brain hemorrhage in the chronic stage after the operation. The length of the elephant trunk prosthesis was 3 cm in the three early patients, and in one of them the elephant trunk could not be utilized due to its insufficient length. In the next three patients, the length was extended to 5 cm, but one of patient experienced an expansion of the aneurysm in the descending aorta due to a graft of insufficient length which could not decompress the aneurysmal wall. Therefore, in the last three patients, the length was further extended to 10 cm, and the second-stage operation was performed uneventfully on the 64th, 9th and 45th day, respectively after the first-stage operation within a continuous hospital stay. Neither expansion of the aneurysm nor thromboembolism was found during the waiting period for any of the second-stage operations. Accordingly, we recommend using a 10 cm elephant trunk prosthesis.

Endovascular stent graft placement for patients with aortic aneurysm and end-organ dysfunction.

Endovascular stent grafts (ESGs) for the treatment of aortic aneurysm is becoming popular because it is less invasiveness for the patient. This new modality seems to be especially useful for treating high risk patients, such as those with end-organ dysfunction. In this study we retrospectively analyzed the results of ESG placement for patients with renal or hepatic dysfunction. From January 1996 to December 1997, six patients with end-organ dysfunction (two with descending thoracic aneurysm and four with abdominal aneurysm) underwent ESG placement. Five of these patients had renal dysfunction, with serum creatinine levels of 2.0 mg/dl or greater, and the remaining patient had hepatic dysfunction with a prothrombin time less than 60%. One of the patients also had severe atherosclerotic disease with a history of multiple brain infarctions. All the patients received custom made endovascular spiral Z stents covered with a woven Dacron (DuPont Co., Wilmington, DE) graft, which was delivered via a femoral artery under local anesthesia. None of the patients showed significant changes in renal or hepatic function after the procedure. None of the five patients with renal dysfunction needed hemodialysis after ESG placement, although the mean preoperative level of serum creatinine and blood urea nitrogen was 3.4 mg/dl and 42.0 mg/ dl, respectively. All the patients left the recovery room on postoperative day 1. These results indicated that endovascular stent graft placement is extremely useful in the treatment of aortic aneurysm patients with end-organ dysfunction.

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses.

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.