Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L.

Stereocontrolled syntheses of model compounds related to a category of the major antigenic epitope against anti-bupleurum 2IIc/PG-1-IgG from an anti-ulcer pectic polysaccharide are described. Glycosylation of the glucuronic acid donors methyl(2,3-di-O-benzoyl-4-O-methyl-alpha-D-glucopyranosyl trichloroacetimidate)uronate and methyl (2,3-di-O-benzoyl-4-O-methyl-beta-D-glucopyranosyl)uronate-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with the common acceptor 2-(trimethylsilyl)ethyl 2,3,4-tri-O-benzyl-beta-D-galactopyranoside in the presence of trimethylsilyl triflate (TMSOTf) gave the desired di- and trisaccharide derivatives. Furthermore the products were transformed into the oligo-valent clustering saccharides, N,N',N"-tri-(5-[4-O-methyl-beta-D-glucopyranosyluronic acid-(1-->6)-beta-D-galactopyranosyloxy]pentylcarbonylaminoethyl)-1,3,5-benzenetriamide and N,N',N"-tri-(5-[4-O-methyl-beta-D-glucopyranosyluronic acid (1-->6)-beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranosyloxy]pentylcarbonylaminoethyl)-1,3,5-benzenetriamide.

Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription.

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

[Hemorheological effects of autologous blood storage before surgery for cardiac valvular diseases].

The hemorheological effects of autologous blood storage with or without the use of erythropoietin were examined before surgery for valvular disease. There was no rheological difference between patients with aortic (16 cases) or mitral (10 cases) valve disease. Before storage, the levels of hematocrit, whole blood viscosity, and especially coefficient of rheology, were lower (p < 0.05) in the blood stored with erythropoietin, but this difference disappeared after storage. The plasma viscosity of both groups did not change before and after storage. The viscosity of blood was equalized after the storage of blood, irrespective of the use of erythropoietin.

Hyperthermia for bone and soft tissue sarcoma: relationship between computerized tomographic and histological findings.

Between 1983 and 1992, 38 patients with 44 tumors of bone and soft tissue sarcoma were treated by hyperthermia combined with radiotherapy or chemotherapy. The overall response rate in this series was 48% (21/44). The average time for heating to 42 degrees C and the average maximum temperature did not correlate with the local effect. The percentage of the low density area on CT images and the average maximum temperature were well correlated. When 100% necrosis was evaluated as CR and up to 50% necrosis as PR, the local response rate was elevated from 42% to > 65%. Bone and soft tissue sarcomas are good targets for hyperthermia combined with radiotherapy and chemotherapy. Some tumors indicated for this study were too large to heat with the present heating apparatus. Percent low density and percent necrosis were correlated to some degree, but adoption of this phenomenon as a criterion for evaluation should be considered carefully.

[Consideration on transfusion in urologic surgery--a retrospective study on homologous and autologous transfusions].

To obtain data for promoting autologous transfusion in urologic surgery, the cases of perioperative homologous transfusions in the past 5 years are reviewed. Although the mean perioperative homologous blood transfusion rate was not so high (10.2%), the rate in common urological surgery was comparatively high (eg. radical nephrectomy 39.4%, simple or palliative nephrectomy 21.6%, total nephroureterectomy 33.3%, renal allotransplantation 82.8%, total cystectomy 96.2%). Mean homologous transfusion volume in these surveys was 982 ml. Two successful cases of autologous blood transfusion by the predeposit method or intraoperative collecting method are presented. Transfusion-associated adverse effects, especially viral infection and immunosuppression, were discussed and the value of autotransfusion was stressed.

Local effect of hyperthermia for superficial and shallow-seated tumors.

In approximately seven years, 134 patients with 161 tumors were treated by hyperthermia combined with radiation or chemotherapy at our department. The primary tumors were breast cancer, head and neck cancer, and soft tissue tumors in most patients. Adenocarcinoma was the most frequent, followed by squamous cell carcinoma and soft tissue sarcoma. The local response rates for primary inoperable advanced, metastatic, and local recurrence of breast cancer were 88% (7/8), 50% (10/15), and 86% (18/21), respectively. The local response rate of 39 tumors of neck lymph nodes was 49% (19/39). A total of 26 tumors of bone and soft tissue were treated. Five tumors showed CR and six PR, for a total response rate of 42%. Among 20 patients with malignant melanoma, CR and PR were 25% (5/20) and 30% (6/20), respectively. The local response rate for all patients with superficial and shallow-seated tumors was 58% (94/161). In some tumors classified as showing NR, complete disappearance of tumor cells was demonstrated by a post-treatment histological examination. The efficacy of hyperthermia, when evaluated solely on the basis of tumor size, is likely to be underestimated.

[Autotransfusion in urological surgery--experience with transurethral resection].

With the purpose to avoid the risk of homologous transfusion, autologous blood transfusion was performed in 26 patients who underwent transurethral resection of the prostate (TUR-P). Autologous blood (200-400 ml/time, 1-3 times, total 200-800 ml, mean 381 ml) was predeposited 4 days to 3 weeks prior to elected TUR-P and was transfused during or just after the operation. Since the start of this program in February 1988, homologous transfusion rate was decreased to 13.1%, whereas it has been 22.3% in 203 cases between January 1984 and January 1988. The mean hemoglobin level fell to 81.6% of the predeposition level. However, the hemoglobin level recovered to 90.1% one month after operation. The circulatory condition of the patients with autologous transfusions was stabler than that of the patients with homologous transfusion or no transfusion. No clinical hemostatic problems occurred. In cases with mild to moderate volume resection of the prostate, this autologous blood transfusion is recommended.

Protective effects of glutathione against lipid peroxidation in chronically iron-loaded mice.

To elucidate the protective effects of glutathione against iron-induced peroxidative injury, changes in the hepatic glutathione metabolism were studied in chronically iron-loaded mice. When the diets of the mice were supplemented with carbonyl iron, iron deposition occurred primarily in the parenchymal cells of the liver. In addition, expiratory ethane production was elevated, suggesting an enhancement in lipid peroxidation. In iron-loaded mice, the total hepatic glutathione contents were higher (6.21 +/- 0.53 mumol/g wet wt.) than in control mice (4.61 +/- 0.31 mumol/g wet wt.), primarily due to an increase in the reduced glutathione contents. The value of oxidized glutathione was also higher (98.5 +/- 8.1 nmol/g wet wt.) than in the controls (60.8 +/- 9.5 nmol/g wet wt.), and the ratio of oxidized glutathione to total glutathione increased. The excretion rate of glutathione from the hepatocytes in iron-loaded mice also increased. These observations suggest that chronic iron-loading of mice stimulates lipid peroxidation and oxidation of glutathione and that peroxidized molecules may be catabolized using reduced glutathione.

[Hyperthermia in bone and soft tissue tumors].

Local response of hyperthermia for soft tissue and bone tumors was investigated. Ten tumors were superficial tumors and 16 were deep seated tumors; 9 tumors were malignant fibrous histiocytoma, 5 were liposarcoma, 4 were neurogenic and 3 were myogenic sarcoma. The other five tumors were an angiosarcoma, a malignant mesenchymoma, an Ewing's sarcoma, a chordoma and an osteosarcoma. Some 23 tumors were heated in combination with radiation therapy, and 3 were combined with arterial infusion of ADR. Four of 10 superficial tumors disappeared (CR), and, 2 of 10 signified PR. Only one of 16 deep seated tumors showed CR, 3 were PR and 12 showed no response. But 4 of 12 tumors without regression in tumor volume indicated coagulation necrosis owing to histological examinations, and 5 of 12 were regarded as the same response from hypodensity area with CT examination after hyperthermia. Local response rate of of superficial tumors was 60% and that of deep-seated tumors was 81.4%.

Results of a non-controlled trial of hyperthermia combined with radiation for superficial tumours.

Fifty-four patients with 65 superficial malignant lesions were treated by local hyperthermia combined with radiation therapy at the National Cancer Center Hospital, Tokyo. Hyperthermia was delivered with an Aloka Model HMS-020 (2450 MHz) or with a horn-type applicator of BSD-1000 (80-90 MHz). Relatively small tumours, those less than 4 cm in thickness, were treated by using 2450 MHz while 80-90 MHz delivered through the horn-type applicator was used for tumours exceeding 5 cm in thickness. The radiation dose was 4 Gy twice a week or 2 Gy five times a week, the total dose being 40-60 Gy. A total of six to 10 hyperthermia treatments ranging from 40 to 60 min each, with the tumour heated to more than 42.5 degrees C, were given twice a week within 1 h following radiation therapy. Complete response was achieved in 16 of the 30 patients (55 per cent) treated with the 2450 MHz microwave, and partial response in seven others (23 per cent). Tumours treated with the BSD-1000 achieved complete response in 10 out of 33 patients (30 per cent) and partial response in nine others (27 per cent). In five out of nine patients classified as partial responders, however, complete disappearance of tumour cells was noted by post-treatment histological examination. Complete plus partial response rates were thus essentially the same with the Aloka HMS-020 and the BSD-1000, though the rate of complete response was apparently higher with the Aloka unit, probably because it was used on smaller tumours.

[Clinical use of combined radiation and hyperthermia therapy at the National Cancer Center Hospital].

At the National Cancer Center Hospital, Tokyo, hyperthermia combined with radiation was started in February 1982. At first BSD-1000 was introduced into clinical practice, followed by Thermotron RF-8 which utilizes radio frequency and then Aloka HMS-020 which utilizes microwaves. This study involved a total of 116 evaluable patients (128 lesions) from a series of patients treated by hyperthermia during a 4-year period up to March 1986. The sites of the tumors were the lower extremities in 7 patients, upper abdomen in 18, pelvic cavity in 18 patients and superficial in 82. Among 82 superficial lesions 34 were classified as showing CR and another 23 as showing PR, with a response rate of 69.5%. In 7 patients with tumors of the lower extremities none was classified as having CR or PR, the response rate being 0%. Eighteen upper abdominal tumors showed a response rate of 16.7%, with 1 of them being categorized as showing CR and 2 as showing PR. Eighteen pelvic tumors showed a response rate of 27.8%, with 1 of them being categorized as showing CR and 4 as showing PR. In 3 patients with tumors of thoracic cavity none was classified as having CR or PR, the response rate being 0%.

Combined 1H and 31P nuclear magnetic resonance spectroscopic studies of bicuculline-induced seizures in vivo.

Using a 1.89-Tesla spectrometer, 1H and 31P nuclear magnetic resonance spectra were acquired from the brains of paralyzed rabbits ventilated with 30% oxygen in nitrous oxide. Intracellular pH and changes in lactate concentration in the cerebrum were monitored by nuclear magnetic resonance methods during and after bicuculline-induced seizures, together with the electroencephalogram, heart rate, and arterial blood pressure. During seizures lasting more than an hour, cerebral intracellular pH became acidic, the cerebral lactate level rose rapidly, and both changes persisted as long as 2 hours without signs of recovery. After less prolonged seizures, lactate elevations were no less persistent, despite nearly complete recovery of intracellular pH and the electroencephalogram.

Biliary and urinary excretion rates and serum concentration changes of four bilirubin photoproducts in Gunn rats during total darkness and low or high illumination.

On cycled exposure of Gunn rats to total darkness and low and high illumination, biliary excretion rates of (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin increased up to approx. 10-fold from the mean basal values of 1.2 and 0.2 microgram/h to the mean maximum values of 25.2 and 4.2 micrograms/h respectively, and at the same time those of (EE)-bilirubin and (EE)-cyclobilirubin also increased, but at very much lower rates than those of the first-mentioned two. During the low illumination only (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin appeared in the urine; during the high illumination (EE)-bilirubin and (EE)-cyclobilirubin also appeared, showing a similar excretion pattern to that observed in the bile, but the total urinary excretion rates were lower than the total biliary excretion rates. The serum bilirubin concentrations fell gradually to lower values, accompanied by an increment in (EZ)- and (ZE)-bilirubin, but (EZ)-cyclobilirubin was not detected. It is concluded that during phototherapy the predominant pathway for the removal of bilirubin from the body in the Gunn rat is by biliary excretion of the geometric photoisomers (EZ)- and (ZE)-bilirubin, derived from Z----E isomerization, and the structural photoisomer (EZ)-cyclobilirubin, formed from intramolecular endo-vinyl cyclization.

Significance of the endo-vinyl group of bilirubin in photochemical reactions.

In photochemical experiments on bilirubin III alpha (no endo-vinyl group), IX alpha (one endo-vinyl group) and XIII alpha (two endo-vinyl groups) and in the photochemical, thermal and catalytical reversion of their photoproducts under anaerobic conditions, much more instability and complexity of photoproducts of bilirubin XIII alpha were observed than for those of bilirubin IX alpha or III alpha. On the basis of present and previous results of photochemical experiments in vitro and the fact that large amounts of (EZ)-cyclobilirubin IX alpha appear in the bile during phototherapy of neonatal hyperbilirubinaemia [Onishi, Kawade, Itoh, Isobe & Sugiyama (1980) Biochem. J. 190, 527-532], it is concluded that the endo-vinyl group plays a crucial role in the photochemical reaction of bilirubin IX alpha. On reversed-phase high-pressure liquid chromatography of photoisomers, it was found that the retention times of geometric isomers and E-cyclized structural isomers were shortened compared with those of Z-isomer and E-isomer, respectively, as precursor substances.

Structure and thermal interconversion of cyclobilirubin IX alpha.

One of the two main photoproducts in bilirubin metabolism during phototherapy in neonatal hyperbilirubinaemia is (EZ)-cyclobilirubin. However, it has not yet been possible to come to a final conclusion as to its chemical structure, despite the fact that much effort has been expended on the problem. The present paper demonstrates that (EZ)-cyclobilirubin is formed by the intramolecular cyclization of the C-3-vinyl group with the position at C-7 rather than at C-6, without delta-lactone-ring formation. The evidence comes from 13C-n.m.r. spectra, which indicate that an oxygen-bound quaternary carbon atom is not present, and from 1H-n.m.r. spectra, which indicate that the orientation of the methyl group at C-2 is equatorial; these findings are supported by mass spectra. The existence of both an epimeric relationship at C-7 between (EE)- and (EZ)-cyclobilirubins A and B and of steric isomers of the hydrogen atom and methyl group at C-2 is supported by the fact that the methyl-group protons at C-2 and C-7 are observed as a paired signal in 1H-n.m.r. spectra, and that new signals at C-7, C-2 and C-3 beta appear in 13C-n.m.r. spectra, that mass spectra of (EZ)-cyclobilirubins A and B are extremely similar and that, furthermore, thermal interconversion between (EE)- and (EZ)-cyclobilirubins A and B is observed.

31P nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells.

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2-deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.