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Quenchbody (Q-body) is a novel fluorescent biosensor based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. In order to develop a method using Q-body for the quantitative determination of deoxynivalenol (DON), a trichothecene mycotoxin produced by some Fusarium species, anti-DON Q-body was synthesized from the sequence information of a monoclonal antibody specific to DON. When the purified anti-DON Q-body was mixed with DON, a dose-dependent increase in the fluorescence intensity was observed and the detection range was between 0.0003 and 3 mg L(-1). The coefficients of variation were 7.9% at 0.003 mg L(-1), 5.0% at 0.03 mg L(-1) and 13.7% at 0.3 mg L(-1), respectively. The limit of detection was 0.006 mg L(-1) for DON in wheat. The Q-body showed an antigen-dependent fluorescence enhancement even in the presence of wheat extracts. To validate the analytical method using Q-body, a spike-and-recovery experiment was performed using four spiked wheat samples. The recoveries were in the range of 94.9-100.2%. The concentrations of DON in twenty-one naturally contaminated wheat samples were quantitated by the Q-body method, LC-MS/MS and an immunochromatographic assay kit. The LC-MS/MS analysis showed that the levels of DON contamination in the samples were between 0.001 and 2.68 mg kg(-1). The concentrations of DON quantitated by LC-MS/MS were more strongly correlated with those using the Q-body method (R(2) = 0.9760) than the immunochromatographic assay kit (R(2) = 0.8824). These data indicate that the Q-body system for the determination of DON in wheat samples was successfully developed and Q-body is expected to have a range of applications in the field of food safety., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study was designed to clarify the influence of long-term enteral nutrition (EN) on the pharmacokinetics of digoxin. Rats were fed EN diets (semi-digested, digested, and elemental) for 4 weeks, then digoxin (0.05 mg/kg) was administered orally. The AUC(0-∞) and k(a) of digoxin were significantly reduced in the semi-digested diet group versus the control, while the AUC(0-∞) was significantly increased in the digested and elemental diet groups. The mRNA level of Slco1a4 was significantly reduced at the upper small intestine in all EN groups. Further, the expression levels of P-glycoprotein (P-gp) protein and Abcb1a mRNA were increased at the same site in all EN groups, and the increases were significant in the elemental diet group. Cyp3a2 protein and mRNA expressions were significantly reduced in the liver in the digested and elemental diet groups. Abcb1a mRNA was also significantly reduced in the kidney in these groups. These results indicate that the absorption kinetics at the small intestine is influenced by semi-digested diet, and the elimination kinetics in the liver and kidney are influenced by digested and elemental diet. Semi-digested diet also altered digoxin pharmacokinetics in humans. Thus, the effect of long-term EN on digoxin pharmacokinetics depended on the dietary components.

Objective: Intravenous tissue plasminogen activator (IV tPA) is an approved treatment for acute ischemic stroke. However, the effects of decompressive craniectomy (DC) after IV tPA administration for ischemic stroke are still largely unknown. The aim of this study was to investigate the safety and outcomes of DC after IV tPA administration., Methods: We retrospectively reviewed patients who underwent DC for malignant hemispheric infarction. We compared 20 patients who underwent DC after IV tPA administration with another 20 patients who underwent DC without prior IV tPA administration., Results: The patient characteristics did not differ between the DC patients with and without prior IV tPA administration. New intracranial bleeding or worsening of pre-existing ICH occurred in two patients (10%) in each group. Furthermore, the rates of an mRS score of 4-6, 5 or 6, and 6 did not differ significantly between the two groups., Conclusion: DC may be a safe and useful surgical procedure for space-occupying edema after IV tPA administration for acute stroke., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Oysters are one of foodstuffs containing a relatively high amount of cadmium. Here we report on establishment of an immunochromatographic assay (ICA) method of cadmium levels in oysters. Cadmium was extracted with 0.l mol L(-1) HCl from oysters and cleaned up from other metals by the use of an anion-exchange column. The behavior of five metals Mn, Fe, Cu, Zn, and Cd was monitored at each step of extraction and clean-up procedure for the ICA method in an inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results revealed that a simple extraction method with the HCl solution was efficient enough to extract almost all of cadmium from oysters. Clean-up with an anion-exchange column presented almost no loss of cadmium adsorbed on the column and an efficient removal of metals other than cadmium. When a spiked recovery test was performed in the ICA method, the recovery ranged from 98% to 112% with relative standard deviations between 5.9% and 9.2%. The measured values of cadmium in various oyster samples in the ICA method were favorably correlated with those in ICP-MS analysis (r(2)=0.97). Overall results indicate that the ICA method established in the present study is an adequate and reliable detection method for cadmium levels in oysters., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Unlabelled: 22-Oxacalcitriol ameliorates high-turnover bone and marked osteitis fibrosa in rats with slowly progressive nephritis., Background: 22-Oxacalcitriol (OCT) is a unique vitamin D analogue with less calcemic activity than calcitriol, and it effectively suppresses parathyroid hormone (PTH) secretion in uremic rats. This study was performed to examine the long-term effect of intravenously administered OCT on high-turnover bone disease in model rats of slowly progressive renal failure., Methods: Slowly progressive renal failure rats were made by a single injection of glycopeptide isolated from rat renal cortical tissues. At 250 days, glycopeptide-induced nephritis (GN) rats were divided into three groups with the same levels of serum creatinine and PTH, and they received either OCT (0.03 or 0.15 microg/kg body wt) or vehicle given intravenously three times per week for 15 weeks., Results: Renal function of GN rats deteriorated very slowly but progressively, as assessed by the increase of serum creatinine concentration. At sacrifice, serum PTH levels, bone formation markers, bone resorption markers, and fibrosis volume were significantly elevated in vehicle-treated GN rats compared with those of sham-operated rats, suggesting the development of high-turnover bone disease with osteitis fibrosa. In contrast, in the GN-OCT 0.15 microg/kg group, these high PTH levels and high-turnover bone and fibrosis were significantly decreased. Such amelioration of bone abnormalities by OCT was not accompanied by either hypercalcemia or further deterioration of renal function., Conclusions: These data indicate that OCT may be a useful and safe agent not only for the suppression of PTH, but also for the amelioration of osteitis fibrosa and high-turnover bone without causing hypercalcemia in chronic dialysis patients.

We have investigated the effect of clonidine on ketamine-induced norepinephrine release from the medial prefrontal cortex in rats using microdialysis. Twenty-one male Wistar rats weighing 200-300 g were allocated randomly to one of four groups: i.p. injection of ketamine 100 mg kg-1 with clonidine 0 (saline: group C0, n = 6), 3 (group C3, n = 5), 30 (group C30, n = 5) and 300 micrograms kg-1 (group C300, n = 5). As reported previously, ketamine increases norepinephrine release. In groups C0 and C3, marked increases in norepinephrine release were observed with maximum values of mean 483 (SEM 55)% and 412 (53)% compared with basal values, respectively. Although significant increases in norepinephrine release were also observed (276 (43)%) in group C30, they were significantly lower than those in groups C0 and C3 (P < 0.01 and P < 0.05, respectively). In group C300, there was a significant reduction in norepinephrine release (62 (13)%) compared with basal and the three other groups (P < 0.01). This inhibitory effect of clonidine on norepinephrine may be related to reduction in undesirable emergence reactions after ketamine anaesthesia.

The inhibitory effects of vitamins A and K toward P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined in the reconstituted system containing the microsomal fraction prepared from the recombinant Saccharomyces cerevisiae cells producing rat P4501A1 and yeast NADPH-P450 reductase. On vitamins A, all-trans-retinol, all-trans-retinal, all-trans-retinoic acid and retinol-palmitate showed competitive inhibition with K(i) values of 0.068, 0.079, 2.6 and 2.0 microM, respectively. Judging from the K(i) values, the inhibitory effects of those vitamins A appear to have physiological significance on the basis of their contents in liver, lung and kidney. On vitamins K, vitamin K(1) showed competitive inhibition with K(i) value of 24 microM, while vitamin K(2) showed noncompetitive inhibition with K(i) value of 60 microM. Judging from these K(i) values together with the contents of these vitamins K in liver, the inhibitory effects of the vitamins K are not as significant as those of vitamins A. These results suggest that the ingestion of enough amounts of vitamins A from foods might lead to the inhibition of the activity of P4501A1 which is known to be induced by smoking, drugs such as omeprazole and lansoprazole, and environmental pollutants like dioxins., (Copyright 1999 Academic Press.)

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains., (Copyright 1999 Academic Press.)

A new xenograft model of multiple myeloma (MM), where growth is strongly regulated by interleukin-6 (IL-6), was established in severe combined immunodeficiency (SCID) mice. In this model, endogenous IL-6 from SCID mice was ineffective at eliciting growth of the established human MM cell line KPMM2; these cells achieved autonomous growth through their autocrine secretion of IL-6. The etiopathology in this disease model is consistent with that of human MM. When greater than 3 x 10(6) KPMM2 cells were injected intravenously (IV), tumors developed in all mice and were predominantly localized in their bone marrow. Tumors were also apparent in the lymph nodes, but absent from other organs. Immunostaining of cell surface antigen (CD38) showed that more than 40% of bone marrow cells in femur were of myeloma origin in the advanced stage of tumor progression (day 37). Histologic analysis of these mice show that bone marrow was largely occupied by plasmablastic cells and bones had developed osteolytic lesions at multiple sites. Concurrently, there was a decrease in bone density throughout the body and a significant increase in ionized plasma calcium. M-protein was detected in the serum within 10 days after transplantation, which correlated with the tumor progression. Between 30 and 40 days after the transplantation, mice presented with a rapid and severe loss of body weight, hind leg paralysis, and fatigue. Subsequently, the mice died within a week. A single IV injection of 0.2 mg humanized anti-IL-6 receptor antibody (hPM1) into mice on the day after tumor transplantation substantially suppressed the elevation of serum M-protein and development of the tumor-associated abnormalities and significantly increased in the life span of tumor-bearing mice. Our data show the usefulness of this model to analyze the pathologic role of IL-6 in MM and the efficacy of targeting the IL-6 receptor in IL-6-dependent KPMM2 cells.

To elucidate the effect of interleukin-6 (IL-6) on bone and bone marrow (BM), human IL-6 transgenic mice (hIL-6 tgm) were produced. Their bone and BM were examined histologically, radiologically, histomorphometrically, and hematologically on a temporal basis. hIL-6 tgm showed histologically evident neutrophilia in BM. Increase in precursors of granulocytes and monocytes in hIL-6 tgm was demonstrated by an assay for colony forming unit in culture (CFU-C) of BM cells. Decrease in osteoblasts and osteoid and suppression of primary spongiosa formation were predominantly observed in hIL-6 tgm at 14 weeks old, the terminal stage of life for hIL-6 tgm. An assay for colony forming unit in fibroblastic (CFU-F) of BM cells revealed a decrease in osteoblast precursor (with regard to alkaline phosphatase-positive colonies) in hIL-6 tgm at 15 weeks old. Histomorphometry demonstrated a decrease of both osteoclast number and bone resorption in hIL-6 tgm. These results suggested that enhanced granulocytic hematopoiesis, suppressed bone turnover, and alteration of cellular population in stromal cells in BM occurred in hIL-6 tgm. Thus we provide new findings that facilitate understanding of cellular interrelationships among hematopoietic cells, osteoblasts, and osteoclasts mediated by stromal cells in BM.

Splenic stromal cells (CF-1 cells) were established from a mouse administered recombinant human granulocyte colony-stimulating factor (rG-CSF) to clarify the mechanism of splenic extramedullary hematopoiesis induced by the factor. The cells were negative for alkaline phosphatase, factor VIII-related antigen, mac I, and phagocytosis. They were positive for acid phosphatase, collagen type I, collagen type III, and fibronectin. CF-1 cells were not converted to adipocytes in a confluent culture with 10(-6) mol/L hydrocortisone. [35S]rG-CSF bound to CF-1 cells specifically in the growth phase but not in the resting phase. The CF-1 cells had greater colony-stimulating activities than the normal splenic stromal cells. When CF-1 cells were added to bone marrow cells in the spleen colony-forming cells (CFU-S) assay, the number of colonies in the spleen increased between 1.4 and 1.8 times the control without these stromal cells. On the other hand, the normal splenic stromal cells had no effect on increasing the number of CFU-S colonies. Therefore, these data suggest that a factor-dependent hematopoietic microenvironment is generated in the spleen by rG-CSF, and the stromal cells that have the hematopoietic potency become dominant in splenic extramedullary hematopoiesis induced by rG-CSF.

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.

The linoleic acid hydroperoxide obtained by enzymatic peroxidation of linoleic acid was found to react with thiobarbituric acid to yield a red pigment. The optimum pH for the reaction was found to be 4.0. In the early stages of peroxidation of linoleic acid, thiobarbituric acid value, the amount of conjugated diene, oxygen consumption, and peroxide value were in parallel with one another. The data were compared with those on peroxidation of linolenic acid and arachidonic acid.

To approach the mechanism of degeneration of the retina in retinopathy of prematurity, a model experiment using chick embryo was carried out. Upon exposing chick embryo at various stages to a high concentration of oxygen at 2 atm pressure, lipoperoxide levels of both the blood and the retina were elevated. The change in lipoperoxide level of the liver was not significant, except for a slight increase at the 9th day of incubation. Upon exposing chick embryo of the 14th day to high concentration of oxygen at ambient pressure, lipoperoxide levels of both the blood and the retina were elevated at 6 h and 12 h of exposure, but significant change was not observed in the lipoperoxide level of the liver.

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.