Preparation and root growth-modulatory activity of N-substituted 2-acetylamino-2-ethoxycarbonyl-3-(2-furyl)propanamides.

N-Substituted 2-acetylamino-2-ethoxycarbonyl-3-(2-furyl)propanamides (8) were synthesized through the reaction of amines (13) with 2-acetylamino-2-ethoxycarbonyl-3-(2-furyl)propanoic acid (3b), which was prepared via condensation of 2-(bromomethyl)furan (10b) with diethyl acetamidomalonate, followed by partial hydrolysis of the resultant diethyl ester (3a) in the presence of barium hydroxide. However, bulky amines such as tert-butylamine or 2-trifluoromethylaniline did not afford the corresponding diamides (8). The biological activity of the prepared diamides (8) as root growth modulators was examined by germination assay using rape and leek seeds. N-(5-Bromo-2-thiazolyl)- and N-(4-chloro-2-benzothiazolyl)-2-acetylamino-2-ethoxycarbonyl-3-(2-furyl)propanamides (8h, i) both potently inhibited the root growth of rape seedlings, but were less effective in the case of leek seeds. The herbicide 2,4-dichlorophenoxyacetic acid completely inhibited root growth in both cases.

Effect of lipase activities of Propionibacterium granulosum and Propionibacterium acnes.

We studied the lipase activities of Propionibacterium granulosum, P. acnes and the suppression of these activities by Jumi-haidoku-to (JHT). Lipase activity of P. acnes biotype III (BIII) was strongest, while that of P. granulosum was faintly expressed. Compared with the control medium, the production of propionic and butyric acids was suppressed by all the tested mediums combined with JHT. The decrease in these acids produced by JHT was significantly higher in P. granulosum than in P. acnes. Although P. acnes BIII may produce a strong effect on acne, the presence of P. granulosum should not be ignored. Further research is required on the correlation between P. acnes and P. granulosum.

Relationship between Propionibacterium acnes biotypes and Jumi-haidoku-to.

We examined the relationship between Propionibacterium acnes biotypes and Jumi-haidoku-to (JHT). In all the P. acnes strains tested, the production of propionic acid (PA) and butyric acid (BA) was suppressed in a medium containing 1 mg/ml JHT compared with the control medium without JHT. There were no significant differences in the rates of decreased PA and BA production between P. acnes biotype 3 (B3) and the other biotypes or between isolates from mild skin rash and more severe skin rash. P. acnes B3 was the most commonly identified biotype. The clinical effects on acne due to the anti-P. acnes lipase activity of JHT did not seem to be influenced by the degree of acne rash or the P. acnes biotype.

Efficacy of Shiunko for the treatment of atopic dermatitis.

Shiunko is a typical Kampo drug made from herbal extracts and used to treat a range of conditions including atopic dermatitis. Shiunko, white petrolatum and 3.5% salt water were applied to the skin of atopic dermatitis patients, and their clinical effectiveness, and the changes in bacterial species and cell numbers on the skin were studied. Staphylococcus aureus was the main species seen but several other coagulase-negative staphylococci were also identified on some patients. Shiunko was clinically effective in four of seven patients (57%) compared with no patients for petrolatum and one of seven (14%) for salt water. Bacterial counts were reduced with Shiunko in four of seven patients compared with one of seven patients (14%) for both petrolatum and salt water. The results suggest that Shiunko may have antibacterial effects on staphylococci, and may be an effective treatment for atopic dermatitis and other skin infections caused mainly by Staphylococcus species.

Comparative study of single-dose and three-day therapy for acute uncomplicated cystitis.

To assess the efficacy and safety of a single-dose therapy for acute uncomplicated cystitis (AUC), we compared 4 treatment regimens in 120 women. Patients eligible for the study were randomly assigned to one of four treatment groups: Ciprofloxacin (CPFX) 200 mg in a single oral dose (group A); 200 mg once daily for 3 days (group B); 200 mg twice daily for 3 day (group C); and cefpodoxime-proxcetil (CPDX-PR) 200 mg once daily for 3 days (group D). The efficacy was evaluated 3 days after the single-dose therapy or at the end of a three-day therapy according to the criteria proposed by the Japanese UTI Committee. The overall clinical efficacy in a total of 107 patients was evaluated to be excellent, moderate, and poor in 72 (67.3%), 35 (31.8%), and 1 (0.9%), respectively. The causative organisms were eradicated in 88.0, 85.2, 85.2, and 82.1% of the patients in groups A, B, C, and D, respectively. Recurrence was identified in 3 (2 in group A and one in group D) of 16 patients who were followed at 2 to 3 weeks after the treatment. No adverse reactions related to the antibiotics were recognized in the study. There were no significant differences in the clinical efficacy or recurrence rate among these four treatment regimens. These results indicate that the single-dose therapy of CPFX is the treatment of choice in women with AUC.

Enzyme immunoassay for a characteristic protein in the animal crude drug lumbricus.

A new method was developed to measure the content of a Lumbricus component in a traditional Chinese medicine (TCM). An antiserum specific to Lumbricus was elicited in a rabbit following immunization with a suspension of Lumbricus fragments. A characteristic antigen protein, 70 kDa, was found in Lumbricus and was purified almost to singleness using a column chromatography series of gel filtration and DEAE-Sepharose. A selected antibody enzyme immunoassay (SAEIA) was developed using the antiserum and the purified 70 kDa protein as a solid-phase antigen. The SAEIA was specific to Lumbricus species, and showed no cross-reaction with any crude drugs other than Lumbricus. This SAEIA detected 70 kDa protein in the amount of 10 ng/ml with excellent reproducibility (coefficient of variation=3.0%) and an EC50 of 0.24 microg/ml. Using this assay, Lumbricus levels were easily determined in a Lumbricus-based TCM Kazecoll, but not in the control Kazecoll (Kakkonto) prepared without Lumbricus. The SAEIA for 70 kDa protein was simple, accurate, reproducible and may provide a general analytical method for the quality control of Lumbricus-based TCMs.

Effect of evodiamine on catecholamine secretion from bovine adrenal medulla.

The effect of evodiamine on catecholamine secretion from bovine adrenal medulla was investigated. Evodiamine, a bioactive component isolated from dry unripened fruit of Evodia rutaecarpa Bentham, was found to stimulate the secretion of catecholamine from perfused bovine adrenal medulla at a concentration of 10 microM and its effect persisted for at least 30 min. This stimulatory effect of evodiamine was abolished by omission of Ca2+ from the perfusion fluid. Evodiamine (0.1-10 microM) markedly enhanced the secretion of catecholamine from the adrenal medulla induced by acetylcholine (100 microM or high K+(56 mM). The secretion of catecholamine was promptly enhanced by acetylcholine or high K+, but returned to the control level on treatment for 20 min. However, when evodiamine was added to the perfusion fluid after acetylcholine or high K+ stimulation for 10 min, the secretion of catecholamine again increased greatly. These results indicate that evodiamine not only stimulated the secretion of catecholamine from bovine adrenal medulla but also reversed insensitivity of these cells to acetylcholine or high K+ stimulation.

Application of a licorice root specific protein to a general method for the assay of licorice root components in traditional Chinese medicines.

A new fuzzy immunoassay method generally applied to ten Glycyrrhizae Radix (GR) preparations of four different botanical origins was studied. Four kinds of antisera were elicited in rabbits immunized with GRs of different botanical origins. The presence of the characteristic GR protein (GRP) was shown using Western blot analyses and selected antibody enzyme immunoassay (SAEIA) methods. A GRP was isolated from one of the GR specimens which was selected using SAEIA methods. The isolated GRP was heated to reduce its binding activity to an anti-GR serum. A new fuzzy SAEIA method generally applicable for assay of the extract of the ten GR specimens was developed using heat-treated GRP as the solid-phase antigen. The fuzzy SAEIA method was successfully applied for the detection and quantitative analysis of the GR component contained in traditional Chinese medicines.

A general enzyme immunoassay for the licorice root component contained in traditional Chinese medicines.

The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Sinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in competition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component was detected using an enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 micrograms/ml for the assay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component contents in Chinese traditional medicines.

Nonoperative treatment for a ruptured pseudoaneurysm of the celiac trunk: report of a case.

We report the case of a 67-year-old man in whom hemorrhage from a ruptured celiac trunk pseudoaneurysm, which occurred as a consequence of leakage at the site of gastroduodenostomy, was successfully controlled by transcatheter arterial embolization (TAE) with stainless steel coils and N-butyl cyanoacrylate (NBCA). The occurrence of a pseudoaneurysm of the celiac trunk associated with anastomotic leakage is etiologically rare. We compiled reports from the literature on TAE for ruptured aneurysms of the celiac trunk, and compared its therapeutic value with that of surgical treatment. Operative death occurred in 4 of a series of 43 patients with aneurysms of the celiac trunk that were surgically treated (9.3%). In 5 patients with ruptured aneurysms, the operative mortality rate was 40% (2/5). Conversely, while the unsuccessful rate of TAE therapy was 17% (1/6), the mortality rate was nil. The patient whose case is presented here was affected by methicillin-resistant staphylococcus aureus (MRSA) at the site of leakage and in the lung. Under septic conditions such as hemorrhage secondary to pancreatitis, the mortality rate of surgical therapy was 23%-29%, whereas the success rate of TAE therapy was 79% and the mortality rate was 4%. Based on these findings, it is suggested that TAE therapy is a viable alternative to surgery for patients even with ruptured pseudoaneurysms of the celiac trunk.

Development and application of a sandwich enzyme immunoassay for Glycyrrhizae Radix protein (GRP) using monoclonal antibodies.

We have developed mouse monoclonal antibodies (anti-GRP mAb-1-5, all IgG1 sub-isotype mAbs) against Glycyrrhizae Radix protein (GRP), which was recently determined to be a marker protein of Glycyrrhizae Radix (GR). Among these, anti-GRP mAb-1 and 2 were found to recognize different epitopes on the GRP molecule, as demonstrated by ELISA analysis, and were used for the development of a sandwich enzyme immunoassay (SEIA) for GRP in traditional Chinese medicines (TCMs). The SEIA was based on the principle of binding an analyte to anti-GRP mAb2 coated on polystyrene microtiter wells, followed by immunoreaction with biotinylated anti-GRP mAb1 and horseradish peroxidase-streptavidin. The SEIA was specific to GRP in GRs, and showed no cross-reaction with any Leguminosae crude drugs other than GRs. This SEIA detected GRP with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 11.5 ng/well and a detection limit of 0.1 ng/well. The present SEIA was about 10-times more sensitive in detecting GRP than the selected antibody enzyme immunoassay (SAEIA) for GRP previously developed using an antiserum to GR itself. Also, the SEIA has such a low assay background that it allowed us to detect a low concentration of GRP in Kyuki-tyoketsu-in-daiichi-kagen (KTIDK), a TCM consisting of only 2.7% GR. The GRP SEIA was simple, accurate, reproducible and may provide a general analytical method for the quality control of GR-based TCMs.

A new method for the detection and quantitative measurement of the contents of trichosanthes root component composing Chinese traditional medicines.

A new method was developed to estimate the content of Trichosanthes root (TR) component in two Chinese traditional medicines. Characteristic antigens of TR were separated from TR extract using rabbit antiserum specific for TR, a dried root tuber of Trichosanthes kirilowii. Two selected antibody enzyme immunoassay (SAEIA) methods, the SAEIA A for assay of TR extract and the SAEIA B for assay of karasurin A were used as detection methods for the separation of TR antigens. Using several column chromatographies, two kinds of TR antigens, a protein component and a glycan component, were separated from TR extract. A SAEIA C method for assay of TR glycan component was developed using biotinylated second antibody and peroxidase-labeled avidin as the detection method. The SAEIA C was also found applicable for specific assay of TR extract as well as for the contents of TR component present in two Chinese traditional medicines, prescriptions of which contained TR. Content of trichosantin, an abortifacient protein component of T. kirilowii, in both the medicines were also measured by applying the SAEIA B for assay of karasurin A. Little trichosantin was found in either medicine and the reason for this was determined.

Specificities of five kinds of antisera produced against crude drugs, pinella tuber, hoelen, glycyrrhizae radix, trichosanthes root and panax ginseng.

Five kinds of rabbit antisera produced against five kinds of crude drugs, Pinella Tuber, Glycyrrhizae Radix (GR), Trichosanthes Root, Hoelen, and Panax Ginseng, and five kinds of extracts of the same crude drugs were prepared. Specificity of each antiserum was demonstrated by two immunological methods for analyses, the selected antibody enzyme immunoassay (SAEIA) and western blotting, and five crude drug extracts as specimens. Each crude drug extract contained characteristic antigen specific to the corresponding antiserum. Characteristic antigens were suggested as protein components. Characteristic antigen of the crude drug GR was separated from GR extract by three chromatographic procedures and a protein component was separated. The separated protein was successfully applied to develop a SAEIA method applicable for specific assays of both the separated protein and GR extract.

[An experimental study on liver hyperthermia using intraperitoneal hyperthermic perfusion (IPHP)].

We evaluated the efficacy of intraperitoneal hyperthermic perfusion (IPHP) in regional hyperthermia of the liver. In white rabbits, an infusion catheter and a drainage catheter were placed in the Douglas pouch and upper abdominal cavity, respectively. Warm saline (46-48 degrees C) was infused into the abdominal cavity at a flow rate of 30-60 ml/min. The temperature was continuously monitored at seven measuring points including water bath, peritoneal inflow line, abdominal cavity, liver parenchyma, portal vein, rectum and esophagus. Temperature of the inflow line was maintained at 46-48 degrees C throughout the course. Temperature of the abdominal cavity reached 42.0-42.6 degrees C 30 minutes after the start of IPHP. The temperature of the portal vein reached 40.9 degrees C and 41.8 degrees C at 30 and 60 minutes, respectively. Liver parenchymal temperature increased to 41.5 degrees C and 42.1 degrees C at 30 and 60 minutes, respectively, indicating effective heating of the liver. On the other hand, the temperatures of the rectum and esophagus remained less than 39.2 degrees C and 40.4 degrees C throughout the course. Therefore, we consider that IPHP will be a simple and effective method for the performance of liver hyperthermia.

Development and application of an enzyme immunoassay for karasurin A, an effective protein component of Trichosanthes kirilowii Max. var. japonicum Kitam.

A method was developed for specific estimation of the content of a non-enzymatic protein, karasurin A, in fractions taken during the extraction and purification processes from a natural source. Anti-karokon serum was elicited in rabbits immunized with fragments of karokon, a dried root tuber of Trichosanthes kirilowii Max. var. japonicum Kitam. Rabbit antibody specific for karasurin A was identified in anti-karokon serum by the Western blotting method. After separation by SDS-PAGE, protein bands of purified karasurin A and extracted proteins from a medicinal herb which is a karasurin A source were reacted with anti-karokon serum followed by treatment with horseradish peroxidase (HRP)-labeled Fab' of goat anti-rabbit IgG, and then bound HRP-labeled second antibody on protein bands was developed to brown by reaction with a substrate solution of the used enzyme. A novel selected antibody enzyme immunoassay (SAEIA) for karasurin A was developed using selective binding of anti-karasurin A antibody in anti-karokon serum to solid phase karasurin A and HRP-labeled Fab' of the second antibody as the tracer. Specific estimation of the content of karasurin A in several fractions taken during the isolation and purification processes of the protein were possible using the SAEIA method.

Resonance Raman spectra of the intermediates in phototransformation of large phytochrome: deprotonation of the chromophore in the bleached intermediate.

Resonance Raman (RR) scattering from type A large phytochrome of pea was measured at cryogenic as well as ambient temperatures to determine an intermediate in which deprotonation of the chromophore takes place. The RR bands of the red-absorbing (Pr) and far-red-absorbing forms (Pfr) of large pea phytochromes at ambient temperatures are almost the same in their frequencies as those of the intact form reported previously (Mizutani et al., 1991). The RR spectrum of large phytochrome excited at 364 nm at -120 degrees C, where Pr and a photointermediate, I700 (= lumi-R), are trapped, showed a strong band at 1625 cm-1 with a shoulder at 1648 cm-1 in the C C=C stretching region. The shoulder disappeared, and a new band appeared at 1597 cm-1 upon raising the temperature to -80 degrees C, where transformation from I700 to meta-Ra proceeds. The RR spectra remained unchanged until -10 degrees C, indicating that the RR spectra of meta-Rb and meta-Rc are close to that of meta-Ra, and we call them comprehensively the bleached intermediate, Ibl. At ambient temperatures where photo-steady-states among a few species are attained, strong RR bands were observed at 1625 and 1599 cm-1 upon excitation at 364 nm under simultaneous far-red illumination, and the 1599-cm-1 band was appreciably intensified under simultaneous red- instead of far-red illumination. By comparison of these spectra with those at low temperatures, the 1625- and 1599-cm-1 bands were reasonably assigned to Pr and Ibl, respectively. A chemically prepared model of the bleached form, Pbl, also gave a prominent band at 1599 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultraviolet resonance Raman spectra of pea intact, large, and small phytochromes: differences in molecular topography of the red- and far-red-absorbing forms.

Ultraviolet resonance Raman (UV RR) spectra excited at 244 nm were observed for pea intact, large, and small phytochromes at pH 7.8. Raman bands assignable to Trp residues dominated the UV RR spectra. The intensity ratios of Trp W7 doublet bands, I(1358)/I(1342), of all three phytochromes in the red light-absorbing form (Pr) were almost the same as that of an aqueous Trp solution, indicating that most of the six and four Trp residues in the 59-kDa chromophoric and the C-terminal 59-kDa nonchromophoric domains, respectively, reside in hydrophilic microenvironments in Pr. This ratio increased under red light illumination, where photoequilibria are attained between Pr and the far-red-absorbing form (Pfr) for intact and small phytochromes and among Pr, a bleached intermediate (Ibl), and Pfr for large phytochromes. The increase of the intensity ratio was most prominent for small phytochromes. These observations suggest that the microenvironments around some Trp residues become more hydrophobic due to conformational changes induced by phototransformation from Pr to Ibl and that the hydrophobicity increase occurs mainly in the chromophoric domain. Among the six Trp residues in the chromophoric domain, Trp365 and Trp567 are likely candidates for those involved in this hydrophobicity increase. The intensity distribution of the amide I band shows little beta-sheet in both Pr and Pfr of the intact, large, and small phytochromes and indicates that alpha-helices and nonregular structure are less populated in the chromophoric domain than in the N-terminal 6-kDa segment and the C-terminal nonchromophoric domain.

Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation.

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.

High-energy proton beam radiation therapy for gynecologic malignancies. Potential of proton beam as an alternative to brachytherapy.

Fifteen patients with advanced gynecologic malignancies were treated with high-energy proton beam radiation therapy (RT) at the Particle Radiation Medical Science Center (PARMS), Tsukuba University, Japan, from 1983 to 1987. The potential of proton beam RT as an alternative to conventional brachytherapy was evaluated. Except for one local recurrence, 14 of 15 patients were locoregionally controlled for 15 to 57 months. Two-year local control rate and 2-year survival rate were 92.3% and 93.3%, respectively. Two cases of transient, radiation-induced proctitis (neither of which required surgical treatment) were the only complications despite a target dose that exceeded 8000 cGy in most cases. The results suggest that sharply localized, high-dose proton beam RT can produce an antitumor effect equivalent to that of conventional brachytherapy.