Novel biosensor for the rapid measurement of estrogen based on a ligand-receptor interaction.

A bioaffinity sensor was developed aiming at the detection of estrogen. This biosensor system is based on the specific binding of estrogen to its receptor immobilized on a gold disk electrode. The recombinant DNA encoding human estrogen receptor ligand-binding domain was expressed in bacteria using the histidine-tag fusion system. The expression of the fusion protein was under control of a bacteriophage T7 promoter, and the protein was purified under native conditions by affinity chromatography, which is based on a specific interaction between a histidine-tag, located in the N-terminus of the protein, and the Ni(II) chelate adsorbent. The protein was immobilized on an Au-electrode with Ni(II)-mediated chemisorption using a histidine tag and thiol-modified iminodiacetic acid. Cyclic voltammetric measurements showed that the reversible electrochemical reaction of a ferrocyanide/ferricyanide redox couple was suppressed by the presence of estrogen in a concentration-dependent manner. It seems reasonable to suppose that the electrostatic property of the protein layer on the electrode surface was altered by complexation with estrogen. These data suggest that this biosensor is applicable to the evaluation binding activities of the chemicals toward the human estrogen receptor.

Evidence for catalytic cysteine-histidine dyad in chalcone synthase.

Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC). DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS. Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS.

Chalcone and stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different cyclization reactions of the common tetraketide to give different products, naringenin chalcone and resveratrol, respectively. We have previously observed in vitro cross-reaction of CHS and STS overexpressed in Escherichia coli, resveratrol production by CHS and chalcone production by STS. When expressed in eucaryotic cells, or in E. coli as thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction. STS refolded from inclusion bodies also showed reduced cross-reaction. While addition of bovine serum albumin and pH in the reaction were without noticeable effect, addition of glycerol decreased the cross-reaction of CHS likely due to its stabilizing effect on enzyme conformation. These results were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi T. et al., FEBS Lett., 460, 457-4 61 (1999)) that the in vitro cross-reaction of CHS and STS is due to intrinsic capability of these enzymes to catalyze different types of cyclization, which, in turn, is endowed by conformational flexibility of their active sites.

Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G(372)FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P(375)G strongly decreased to give various products in the order CTAL> resveratrol approximately bisnoryangonin>naringenin. As a result, naringenin production (cross-reaction) by STS-P(375)G was close to 30% of resveratrol production. Both G(374)L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G(372)FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.

Effect of the 21-aminosteroid on nuclear factor-kappa B activation of Kupffer cells in endotoxin shock.

BACKGROUND: The 21-aminosteroid (U-74389G) is a nonglucocorticoid steroid that was synthesized to inhibit lipid peroxidation without the glucocorticoid activity. We recently demonstrated that the 21-aminosteroid administered to endotoxin shock mice reduces liver injury and improves the survival rate of mice through inhibition of nuclear factor-kappa B activation in the liver. The study was undertaken to determine whether the 21-aminosteroid could suppress pro-inflammatory gene up-regulation through inhibition of nuclear factor-kappa B activation in Kupffer cells. METHODS: Kupffer cells were isolated from rats by collagenase perfusion followed by pronase digestion. After a lipopolysaccharide addition, each assay was performed for tumor necrosis factor-alpha, interleukin-6, tumor necrosis factor-alpha messenger RNA, nuclear factor-kappa B, and I kappa B proteins. RESULTS: After the lipopolysaccharide addition, Kupffer cells released both tumor necrosis factor-alpha and interleukin-6. The 21-aminosteroid treatment suppressed the release of tumor necrosis factor-alpha in a dose-dependent manner. The 21-aminosteroid also inhibited the increase of tumor necrosis factor-alpha messenger RNA expression and nuclear factor-kappa B activation in Kupffer cells 1 hour and 30 minutes, respectively, after lipopolysaccharide addition. Furthermore, the 21-aminosteroid treatment suppressed the degradation of I kappa B proteins in lipopolysaccharide-stimulated Kupffer cells. CONCLUSIONS: These results suggest that the 21-aminosteroid inhibits release of the tumor necrosis factor-alpha and interleukin-6 from lipopolysaccharide-stimulated Kupffer cells by inhibiting nuclear factor-kappa B activation. This is accomplished by inhibiting I kappa B degradation in endotoxin shock and this may prove useful for the treatment of endotoxin shock.

Effect of lazaroid U-74389G and methylprednisolone on endotoxin-induced shock in mice.

BACKGROUND: Lazaroids are nonglucocorticoid analogs of methylprednisolone with multiple actions. We investigated whether lazaroid U-74389G could attenuate endotoxin-induced liver injury. We hypothesized that U-74389G treatment may protect against hepatic injury by suppressing proinflammatory gene up-regulation through inhibition of activation of nuclear factor kappa B (NF-kappa B). We also compared the efficacy of U-74389G with methylprednisolone in endotoxin-induced liver injury. METHODS: Lipopolysaccharide (Escherichia coli, 30 mg/kg given intraperitoneally) was administered to male ICR mice, and U-74389G (3 mg/kg intraperitoneally) or methylprednisolone (30 mg/kg intravenously) was administered simultaneously. Phosphate-buffered saline solution (0.15 mL intravenously) was administered to mice that served as a control group. RESULTS: U-74389G and methylprednisolone treatment significantly increased survival rates 48 hours after lipopolysaccharide injection and protected against lipopolysaccharide-induced liver injury in vivo, as indicated by the decreased hepatic lipid peroxidation, tumor necrosis factor-alpha, and inducible nitric oxide synthase messenger RNA formation, hepatic enzyme release, and neutrophil infiltration in the liver. U-74389G and methylprednisolone also showed inhibitory effects on NF-kappa B activation in the liver. CONCLUSIONS: These findings suggest that U-74389G can suppress proinflammatory gene up-regulation through inhibition of NF-kappa B activation and that it is a promising new antioxidant drug for the treatment of endotoxin shock.

Effect of the angiotensin-converting enzyme inhibitor enalapril on post-transplant erythrocytosis.

Post-transplant erythrocytosis (PTE) is increasingly recognized as a complication of kidney transplantation. In this study we report the effect of the angiotesin-converting enzyme (ACE) inhibitor enalapril on hematocrit (Ht) and erythropoietin in four patients with PTE. Four renal allograft recipients with Ht greater than 51% were studied. Treatment was initiated with enalapril administered orally at a dose of 2.5 mg/day. All the patients had an increase of hemoglobin (Hb) (17.7 +/- 0.64 g/dl), Ht (54.5 +/- 1.29%) and red blood cell count (RBC) (584 +/- 19.2 x 10(4)/microliter). All patients responded to enalapril in 8 weeks with a significant decrease of Hb, Ht, and RBC. In one patient, the downward trend was more rapid and sustained, and treatment had to be discontinued to prevent the development of anemia. Serum erythropoietin showed normal in all four patients and remained unchanged during the study, even after discontinuation of enalapril treatment. Serum creatinine remained relatively stable throughout the study. These results suggest that PTE may not be dependent upon circulating erythropoietin and that enalapril treatment may be an effective treatment of PTE without renal dysfunction.

Malignant mesothelioma originating in the hepatic falciform ligament: report of a case.

We report herein what to our knowledge is the first documented case of malignant mesothelioma arising from the falciform ligament. A 65-year-old woman was admitted to our department for the evaluation of an epigastric mass. An abdominal computed tomography scan, magnetic resonance imaging, and celiac angiogram showed a tumor originating in the falciform ligament. The tumor was subsequently resected, and pathological examination confirmed a diagnosis of malignant mesothelioma originating in the falciform ligament. A metastatic liver tumor was detected 3 years after this operation and a partial liver resection was performed. She is currently off therapy, and no clinical or radiologic evidence of disease has been found for 1 year since her second operation.

[Study on abuse and neglect of the disabled elderly living at home].

To obtain basic information on elderly abuse and neglect in order to provide for its early discovery and prevention, a questionnaire survey about the present situation regarding abuse and neglect of the disabled elderly living at home was performed on health/medical service/welfare professions. Forty-two cases were analyzed. The major results were as follows; 1. Victims were 13 men and 29 women, approximately 1/3 of whom were in their 70's and another 1/3 in their 80's. Approximately 1/4 of the abusers were either sons or daughters of the victims. 2. Verbal abuse was the most frequent type of abuse with a rate of 69.0%. The percentage of psychological abuse was 61.9, passive neglect was 57.1%, active neglect was 50.0%, physical abuse was 47.6%, passive self-neglect was 28.6%, financial/material exploitation was 23.8%, active self-neglect was 16.7% and the others was 11.9%. The average number of abuse and neglect that an elderly received was 3.5. 3. The main causes of abuse and neglect did not appear to be simple but was complicated by related causes, many of both victims and abusers had elements in their personality, developmental history and interpersonal relationships that over a period of years formed the basis for the problematic behavior. Many abuse or neglect cases arose from caregiving burden of family caregivers or insufficient social systems for support to meet the needs of caring for the elderly. 4. The results suggest that for prevention and countermeasures for elderly abuse, there is an urgent need to arrange for expansion of health and welfare services to reduce burden of caregivers, provide for education of professionals of health/medical service/welfare for early discovery and proper handling of abuse problems, development of a checklist for early discovery, expansion of opportunities of improving care skill for family caregivers and establishment of consultation system for the elderly and caregivers, and organizing emergency shelter for victims of elderly abuse.

Tumor localization studies and surgical treatment in patients with insulinoma.

In the present study, we retrospectively reviewed thirteen consecutive patients with insulinomas including 2 reoperations at our department for insulinomas, from the viewpoint of preoperative localization studies, surgery and long term follow-up. The results of the preoperative localization studies proved to be percutaneous transhepatic portal venous sampling (PTPVS) 6/7, angiography 8/15, ultrasonography (US) 6/11, endoscopic ultrasonography (EUS) 4/4, computed tomography (CT) 3/10, and magnetic resonance imaging (MRI) 2/2. The tumor was visualized by intraoperative ultrasonography (IUS) in 6 of 6 patients (100%). Six patients underwent enucleation, 6 patients underwent distal pancreatectomy, 2 patients underwent subtotal (80%) distal pancreatectomy, and one patient a pylorus preserving pancreaticoduodenectomy (PPPD). Two patients, one of whom belonged to the MEN-I group, underwent reoperations because they had multiple adenomas. The development of iatrogenic diabetes occurred in the case of 3 patients. These results suggest that the use of selected preoperative localization studies (PTPVS and probably EUS) may be very helpful for detecting insulinoma, and that IUS is an essential part of the operative exploration for insulinoma. Our data may further indicate the need for an aggressive surgical procedure in the case of multiple adenoma or insulinoma in MEN-I.

A case of abdominal aortic aneurysm associated with systemic lupus erythematosus.

A case of abdominal aortic aneurysm associated with systemic lupus erythematosus (SLE) is reported. A 45-year-old woman with a 18-year history of SLE was admitted with severe lumbago radiating to the bilateral inguinal region. CT and DSA showed a dumbbell shaped true aneurysm of the abdominal aorta. An aorto-biiliac Y shaped graft replacements was performed. SLE is rarely associated with aneurysm of the great arteries. We could find only 4 reports of abdominal aneurysm associated with SLE. Common features were the young age of the patient, the long term of the systemic disease, and administration of corticosteroid therapy for a relatively long period of time. We speculate that atherosclerosis, hypertension, and corticosteroid may all work in concert, possibly together with aortic wall involvement or vasculitic damage, to produce the rare abdominal aneurysm in SLE.

[Association of participation in health checkups with medical service utilization in a rural town].

Association of participation in health checkups in 1983-94 with medical service utilization in 1989-94 was studied. The study subjects consisted of 1,198 males and 1,247 females aged 30 years or over, who were members of the National Health Insurance in a rural town in Kyoto prefecture. One hundred and four males and 95 females died during 1989-94. Cumulative death rates were higher among both males and females with less participation in checkups than among those with higher participation, however, there was no difference seen for medical service utilization. Regarding subjects alive in 1994, both males and females who participated frequently in checking in 1983-88 also visited medical facilities more frequently and had higher medical costs in 1989-94 than those with less frequent participation. Similar analysis for checkups in 1989-94, showed that only females had that tendency. Among participants in checkups in 1989-94, there were smaller numbers of hypertensives, current smokers, and current drinkers than among non-participants, therefore, the participants are thought to have decreased their risk for chronic diseases. In conclusion, frequent participation in health checkups are thought to increase medical utilization even with a lower prevalence or risk of chronic diseases.

Protective effect of monoclonal antibodies to adhesion molecules on rat liver ischemia-reperfusion injury.

BACKGROUND: The source of reactive oxygen species in the liver remains to be elucidated. The present study was undertaken to determine whether polymorphonuclear neutrophils (PMNs) can contribute to hepatic ischemia-reperfusion injury, and pretreatment with monoclonal antibodies (mAbs) to intercellular adhesion molecule-1 (ICAM-1), lymphocyte function associated antigen-1 (LFA-1), and CD 18 could improve energy metabolism and prolong the viability of the organ. METHODS: Male Wistar rats were used. Rat liver ischemia was induced by clamping blood vessels supplying median and left lateral hepatic lobes. Monoclonal antibodies to ICAM-1, LFA-1, or CD18 were injected intravenously 5 minutes before inducing ischemia. To determine the effect of mAbs on the survival rate, total hepatic ischemia was induced by clamping the hepatic artery, portal vein, and bile duct after making a portafemoral shunt. RESULTS: Although ischemia of the liver for 90 minutes did not permit survival of the animals, pretreatment with mAbs to ICAM-1 plus LFA-1 increased the survival rate to 57%. Pretreatment with mAb to ICAM-1 failed to increase the survival rate. The number of PMNs in the liver increased continually up to 24 hours after reperfusion after 90 minutes of ischemia, and the expression of ICAM-1 was enhanced 4 hours after reperfusion. This is accompanied by a low recovery of hepatic adenosine triphosphate and, on the contrary, a marked increase in lipid-peroxide in the reperfused liver. Pretreatment with mAbs suppressed the infiltration of PMNs and the elevation of lipid peroxide and enhanced the recovery of hepatic adenosine triphosphate 6, 12, or 24 hours after reperfusion. Pretreatment with mAbs also prevented the rise in serum alanine aminotransferase level after reperfusion. CONCLUSIONS: These results suggest that PMNs contribute to ischemia-reperfusion injury in the liver 4 hours and more after reperfusion, and pretreatment with mAbs to adhesion molecules is useful for the prevention of ischemic liver cell injury.

Effect of antileukocyte adhesion molecule antibodies, nitric oxide synthase inhibitor, and corticosteroids on endotoxin shock in mice.

We compared the therapeutic effects of anti-leukocyte adhesion molecule antibodies (mAbs), a nitric oxide (NO) synthase inhibitor (monomethyl-L-arginine, NMLA), and methylprednisolone (MP) on experimental endotoxin-induced shock in mice. Lipopolysaccharide (LPS, 30 mg/kg) was administered to ICR mice intraperitoneally, While 1 mg/kg mAb, 5-20 mg/kg NMLA, or 30 mg/kg MP was administered intravenously. The placebo group received phosphate-buffered saline. The survival rate of the placebo group 48 h after LPS injection was 36%. The administration of anti-CD11a, anti-CD18, anti-lectin cell adhesion molecule-1 (anti-LECAM-1), and MP increased the survival rate to 70, 62, 64, and 100%, respectively; however, NMLA had no significant effect. A FACS analysis revealed that the CD18 expression of granulocytes increased 12-fold within 30 min after LPS administration. MP significantly suppressed its expression. The plasma level of nitrate/nitrite increased from 20 to 260 and 1000 microM 4 and 16 h, respectively, 20 mg/kg NMLA abolished NO production at 4 h, while MP inhibited it for up to 16 h. The hepatic malondialdehyde level increased from 0.50 to 2.46 nmol/mg protein at 4 h. Administration of anti-CD18 and MP reduced the level to 1.80 and 1.41 nmol/mg protein, respectively, whereas NMLA did not affect it. The mAbs and MP were concluded to be useful agents for endotoxin shock. The abolition of NO production had little influence on the hepatic cellular injury associated with endotoxemia.

Activated (HLA-DR+) T-lymphocyte subsets in cervical carcinoma and effects of radiotherapy and immunotherapy with sizofiran on cell-mediated immunity and survival.

A prospective randomized trial on 312 patients with locally advanced cervical carcinoma (FIGO stages IB-IV) was carried out. The 5-year survival in 90 patients treated with radiotherapy and antitumor polysaccharide sizofiran, an extract from the culture broth of Schizophyllum commune Fries, in combination was significantly (P = 0.045) better than that in 82 patients treated with radiotherapy alone. Treatment with sizofiran and 5-fluorouracil in combination improved (P = 0.003) the 5-year survival in 60 patients treated with radiotherapy. In 244 cervical carcinoma patients, the percentage of activated CD8+ (CD8+HLA-DR+) T cells in the CD8+ T-cell subsets in peripheral lymphocytes increased significantly as the disease progressed. A similar tendency was observed in the percentage of activated CD4+ (CD4+HLA-DR+) T cells in the CD4+ T-cell subsets. These immunologic parameters were significantly increased by radiotherapy, but not by surgery. Sizofiran accelerated a recovery in the activated CD8+ T cells in the CD8+ T-cell subsets compared with that of sizofiran nontreated patients after radiotherapy. Our data show that possible immune impairment in cervical carcinoma may be caused by disturbances in cell-mediated immunity, and that sizofiran is an effective immunotherapeutic agent for cervical carcinoma because it stimulates a rapid recovery of the immunologic parameters impaired by radiotherapy.

Clinical application of low calcium peritoneal dialysate.

Low calcium dialysate (LCa) with calcium levels of 2.3 mEq/L was prepared for peritoneal dialysis, and its equilibration was compared with those of conventional dialysates with calcium contents of 3.5 mEq/L and 4.0 mEq/L. The variation in dialysate to plasma concentration ratios of total calcium of conventional dialysates was positive; that of LCa was negative. Mass transfer of calcium per day was positive and normal for calciums of 4.0 mEq/L (31 +/- 51 mg/day) and 3.5 mEq/L (-37 +/- 40 mg/day), compared with a negative balance for LCa (-157 +/- 26 mg/day). The LCa was used in six patients over 6 months. The doses of calcium carbonate and vitamin D also were increased from 1.2 +/- 1.5 to 5.4 +/- 3.3 g/day (p less than 0.05) and from 0.17 +/- 0.2 to 0.38 +/- 0.47 micrograms/day, respectively. Aluminum gel was discontinued. Low calcium dialysate may become the standard dialysate for stable continuous ambulatory peritoneal dialysis patients.

Angiosarcoma of the vagina. A light and electronmicroscopy study.

A 73-year-old woman, whose medical history reported radical hysterectomy and radiotherapy for squamous cell carcinoma of the cervix uteri 20 years earlier, was found to have an angiosarcoma of the vagina. The histological diagnosis was confirmed by immunohistochemical staining of tumor cells for factor VIII-related antigen and ultrastructurally defined vasoformative structures. Primary angiosarcoma of the vagina is quite rare and reported in only two published cases. The present case is a third in general, and the first report confirmed by light microscopy, immunohistochemical, and electronmicroscopy studies.

[Effect of RU 486 on glycogen metabolism in endometrium].

To elucidate the effect of the antiprogesterone steroid RU 486 (RU) on endometrial glycogen metabolism, a dose of 30 mg/kg of the agent was administered to pregnant rats on Day 2 (group 1) or Day 4 (group 2) of pregnancy, and glycogen content, glycogen synthetase (GS) and glycogen phosphorylase (GP) activities in the endometrium were investigated on Day 6. In addition, serum ovarian steroid hormones and the number of implantation sites were evaluated. The glycogen content in the endometrium in group 1 and group 2 decreased significantly (p less than 0.02 and p less than 0.05) compared with the control group. The total GS activity in the endometrium in group 1 decreased significantly (p less than 0.05). On the other hand, the independent GS activity in group 2 increased significantly (p less than 0.05) compared with the control group. The active GP activity in group 1 increased markedly, and the total GP activity increased significantly (p less than 0.05) compared with the control value. In group 2, the active and total GP activities increased significantly (p less than 0.01 and p less than 0.02) compared with the control values. Serum progesterone (P) concentrations in group 1 and group 2 were significantly (p less than 0.05 and p less than 0.05) lower than those in the control groups 2 days after the RU administration, and implantations were significantly (p less than 0.01 and p less than 0.01) inhibited. These results suggest that RU 486 affects endometrial glycogen metabolism, resulting in the prevention of implantation.