Source of N-nitrosodimethylamine in river waters of the upper Tone River basin in Japan.

The presence of N-nitrosodimethylamine (NDMA) in the Hirose River and its tributaries, located in the upper Tone River basin, in the Kanto region of Japan, was investigated. NDMA was detected at high levels in the Arato River, one of the tributaries of the Hirose River, at high concentrations (up to 2,100 ng/L). Due to the confluence of the Arato River, NDMA concentration in the Hirose River increased (up to 61 ng/L). The NDMA in the Arato River was due to industrial discharge from a livestock processing plant located near the river. There were three discharges at the plant, with NDMA concentrations of 78, 11, and 33,000 ng/L. The industrial discharges from the livestock processing plant did not contain significant amounts of NDMA precursors on chloramination. On the other hand, sewage effluent was shown to contain NDMA precursors. The amounts of NDMA precursors in the sewage effluent that were rapidly transformed into NDMA were considered to be lower than those slowly transformed into NDMA.

Removal of selected pharmaceuticals by chlorination, coagulation-sedimentation and powdered activated carbon treatment.

Removal property of nine pharmaceuticals (clofibric acid, diclofenac, fenoprofen, gemfibrozil, ibuprofen, indomethacin, ketoprofen, naproxen and propyphenazone) by chlorination, coagulation-sedimentation and powdered activated carbon treatment was examined by laboratory-scale experiments under the conditions close to actual drinking water treatment processes. Indomethacin and propyphenazone were completely degraded by chlorination within 30 minutes, but others remained around 30% (naproxen and diclofenac) or more than 80% of the initial concentration after 24 hours. A couple of unidentified peaks in a chromatogram of the chlorinated samples suggested the formation of unknown chlorination by-products. Competitive adsorption was observed when the mixed solution of the target pharmaceuticals was subjected to batch adsorption test with powdered activated carbon. Clofibric acid and ibuprofen, which were relatively less hydrophobic among the nine compounds, persisted around 60% of the initial concentration after 3 hours of contact time. Removal performance in actual drinking water treatment would become lower due to existence of other competitive substances in raw water (e.g. natural organic matter). Coagulation-sedimentation using polyaluminium chloride hardly removed most of the pharmaceuticals even under its optimal dose for turbidity removal. It is suggested that the most part of pharmaceuticals in raw water might persist in the course of conventional drinking water treatments.

Roles of long chain fatty acids and carnitine in mitochondrial membrane permeability transition.

Palmitoyl-CoA (Pal-CoA) lowered the respiratory control ratio (RCR), and induced mitochondrial membrane permeability transition (MPT) and cytochrome c (Cyt. c) release from isolated rat liver mitochondria. L-Carnitine suppressed the Pal-CoA-induced dysfunction, MPT, and Cyt. c release of isolated mitochondria. This suppression was inhibited by cephaloridine, an inhibitor of carnitine uptake into mitochondria. Cyclosporin A (CsA), an inhibitor of MPT, and BSA also suppressed the Pal-CoA-induced MPT. In the presence of inorganic phosphate (P(i)), Ca2+-induced MPT was suppressed by BSA, L-carnitine, and chlorpromazine, an inhibitor of phospholipase A2. In the presence of a low concentration of Ca2+, 3,3',5-triiodothyronine, long chain fatty acids, salicylic acid, and diclofenac induced MPT by a mechanism that was suppressed by BSA, L-carnitine, or chlorpromazine. During the incubation of mitochondria on ice, their respiratory competence decreased; L-carnitine and BSA also prevented this decrease. Mitochondrial depolarization in pheochromocytoma PC12 cells was induced by either serum deprivation or arachidonic acid by a mechanism that was suppressed by acetyl-L-carnitine. These results indicate that some MPTs may be regulated by fatty acid metabolism and that the Pal-CoA-induced MPT plays an important role in the induction of apoptosis.

Phosphatidylinositol 4-phosphate 5-kinase Its3 and calcineurin Ppb1 coordinately regulate cytokinesis in fission yeast.

The ppb1(+) gene encodes a fission yeast homologue of the mammalian calcineurin. We have recently shown that Ppb1 is essential for chloride ion homeostasis, and acts antagonistically with Pmk1 mitogen-activated protein kinase pathway. In an attempt to identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature, and isolated a mutant, its3-1. its3(+) was shown to be an essential gene encoding a functional homologue of phosphatidylinositol-4-phosphate 5-kinase (PI(4)P5K). The temperature upshift or addition of FK506 induced marked disorganization of actin patches and dramatic increase in the frequency of septation in the its3-1 mutants but not in the wild-type cells. Expression of a green fluorescent protein-tagged Its3 and the phospholipase Cdelta pleckstrin homology domain indicated plasma membrane localization of PI(4)P5K and phosphatidylinositol 4,5-bisphosphate. These green fluorescent protein-tagged proteins were concentrated at the septum of dividing cells, and the mutant Its3 was no longer localized to the plasma membrane. These data suggest that fission yeast PI(4)P5K Its3 functions coordinately with calcineurin and plays a key role in cytokinesis, and that the plasma membrane localization of Its3 is the crucial event in cytokinesis.

Efficacy of antithrombin replacement therapy in severe early-onset preeclampsia.

It has been suggested that the hypercoagulable state in severe preeclampsia is strongly related to the onset of intrauterine growth retardation (IUGR) through the deterioration of placental circulation. In this study, one of two kinds of anticoagulants, heparin or antithrombin (AT), was given to severe early-onset preeclamptic women (onset before 32 weeks of gestation) with IUGR, and the efficacies in maternal and fetal findings were compared. The mechanism of AT to improve placental circulation was discussed based on our previous study using the culture system of chorionic trophoblastic cells. The mean systolic blood pressure decreased significantly in the AT group (AT concentrate 1,500 IU/d for 7 days, n = 15). Fetal growth was calculated by ultrasonographic measurement, and the weight gain was higher in the AT group than it was in the heparin group. In vitro experiments showed that AT increased the thrombomodulin (TM) antigen on the cell surface and also increased prostaglandin I2 (PGI2) production by cultured trophoblastic cells. This suggests that AT replacement therapy is useful for improving maternal hypertension and fetal findings in severe preeclampsia with IUGR through the increase of TM and PGI2 production in both maternal and placental circulation.

Short-homology-independent illegitimate recombination in Escherichia coli: distinct mechanism from short-homology-dependent illegitimate recombination.

We have shown elsewhere that there is no, or very little, homology at the recombination sites in DNA gyrase-mediated illegitimate recombination in vitro. On the other hand, many reports have indicated that illegitimate recombination takes place between sequences with a short homology. To clarify this contradiction, we analyzed the mechanism of DNA gyrase-mediated illegitimate recombination in vivo, by isolating a temperature-sensitive gyrA mutant (gyrAhr1) that causes spontaneous illegitimate recombination at a higher frequency than that of the wild-type. This mutant also causes spontaneous induction of lambda prophage. It is therefore suggested that the gyrAhr1 mutation induces strand breaks in the chromosome, resulting in the formation of illegitimate recombinants. Analysis of the recombination junctions of lambdabio transducing phages formed spontaneously in the gyrAhr1 mutant revealed that the Escherichia coli bio and lambda recombination sites have an average homologous sequence of only 1.3 base pairs. This is the first indication that homology in vivo is not required for illegitimate recombination. On the other hand, a short homology of 8.4 bp, on average, was found in the junctions of lambdabio transducing phages formed spontaneously in the wild-type bacteria. When the gyrAhr1 mutant was irradiated with UV, short homologies were also detected in the junctions. We concluded that illegitimate recombination, which takes place spontaneously in the gyrAhr1 mutants, is distinguishable from spontaneous recombination in the wild-type and from UV-induced recombination in the mutant with regard to the requirement for short homology. We propose that short-homology-independent illegitimate recombination is mediated by subunit exchange between DNA gyrase, while short-homology-dependent recombination is triggered by double-strand breaks and completed by processing, annealing, and ligation of DNA ends.

The frequencies of diabetic complications in elderly non-insulin dependent diabetic patients in Himeji.

Data on 746 patients with non-insulin-dependent diabetes mellitus (NIDDM) were collected from the Internal Medical Association in Himeji by questionnaire, and the patients were divided into six groups according to the duration of illness. Frequencies of various complications according to the duration of illness and risk factors of complications were compared between men and women. Although the number of male patients was 417, significantly more than the 329 female patients, many female patients were elderly, and the age at initial onset was about 10 years older than that of the male patients. Fasting blood sugar and hemoglobin A1c levels increased with the duration of illness. The female patients showed a greater tendency to suffer from hypertension, hyperlipidemia and obesity than the male patients. There was positive correlation between the incidence of complications and duration of illness. This tendency was more marked in the female patients than in the male patients. Both male and female patients showed a tendency for microangiopathy to appear earlier than macroangiopathy. The increase in the frequency of complications accompanying the increase in the duration of illness was more marked for microangiopathy than for macroangiopathy.

Temporary appearance of a circulating granulocyte-macrophage colony-stimulating factor in lethal murine malaria.

Infection of mice with Plasmodium berghei engendered a temporary appearance of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the serum. The peak of GM-CSF levels was detected at day 2 post-infection, and then gradually decreased. On the other hand, the number of committed stem cells for granulocytes and macrophages (CFU-GM) in bone marrow transiently decreased at day 2 post-infection, and then increased and peaked at day 6 post-infection. When the serum of P. berghei-infected mice was fractionated by gel chromatography on Sephacryl S-300, GM-CSF activity was detected as a single peak with an apparent molecular weight of 64 KDa. GM-CSF was entirely adsorbed to concanavalin A-Sepharose 4B affinity chromatography, and was sensitive to pronase digestion, indicating its glycoprotein nature. These results suggest that the circulating GM-CSF would contribute the increase of granulocyte-macrophage hemopoiesis in the early phase of malaria.

Disposition of CS-670, a novel nonsteroidal anit-inflammatory drug, and its metabolites in healthy human volunteers.

CS-670, a novel nonsteroidal anti-inflammatory drug, is a racemic prodrug. Plasma concentrations and urinary excretion of CS-670 and its metabolites were determined in experimental subjects after oral administration at a single 120 mg dose. CS-670 and four metabolites, the saturated ketone (M-A), unsaturated-alcohol (M-B), cis-alcohol (M-C), and trans-alcohol (M-D), were quantitated by GC-MS. The major metabolites in human plasma were M-B, M-C, and M-D and their terminal half-lives (t1/2) were 0.9, 2.6, and 1.2 h, respectively. The total recovery in the urine was 26% of the dose, but unchanged CS-670 accounted for less than 2% over a 48 h period. In addition, the absolute configurations of the metabolites were examined by HPLC after derivatization with chiral reagents. It was found that the configuration of the propionic acid moiety of the metabolites, M-B, M-C, and M-D, in human plasma, was rapidly inverted from (-)-(R) to the (+)-(S) configuration in stereoselective biotransformation. Furthermore, the configurations of the 1'- and 2'-carbons of M-C and M-D, were found to be (1'R, 2'S) and (1'R, 2'S), respectively. These results show that CS-670 is readily biotransformed by chiral inversion of the 2-arylpropionic acid moiety and stereoselective reduction of the alpha, beta-unsaturated ketone moiety in humans.

The prevalence of diabetic complication of elderly diabetics in Himeji.

Diabetes mellitus has recently markedly increased among elderly patient's diseases. There are no recent epidemiological reports on the relative number of male and female diabetic patients. So, an epidemiological study was performed on 746 Non-Insulin-Dependent Diabetes Mellitus patients, whose data were obtained from members of the Himeji Internal Medicine Association, divided into six groups according to sex and duration of illness. The following results were obtained. 1) The number of male patients was greater by about 20% than that of female patients, while elderly patients accounted for a larger proportion, nd age at onset of disease was about ten years higher in female than in male patients. 2) All indicators of diabetes mellitus became worse with longer duration of illness. 3) There was a correlation between the prevalence of complications and the duration of illness: The prevalence of complications increased in parallel with increasing duration of illness, and this tendency was more marked in female than in male patients. 4) Female patients had a more marked tendency to develop hypertension, hyperlipidemia and obesity than male patients. 5) Microangiopathy generally manifested itself earlier than macroangiopathy, and the increase in the prevalence of angiopathy in accordance with prolonged duration of illness was more marked for microangiopathy than for macroangiopathy. Clinical features of Japanese diabetics are found to be similar to those of Europeans, especially dominant in females. This might be due to the changing life style in japan.

Stereospecific taurine conjugation of the trans-OH metabolite (active metabolite) of CS-670, a new 2-arylpropionic acid nonsteroidal anti-inflammatory drug, in dogs.

CS-670, (+/-)-2-[4-(2-oxocyclohexylidenemethyl)phenyl]propionic acid, is a novel derivative of 2-arylpropionic acid non-steroidal anti-inflammatory drugs (profen NSAIDs). The major urinary metabolite of this drug from dogs was isolated and its chemical structure was determined by MS and NMR spectroscopy. The metabolite was identified as a taurine conjugate of the trans-OH form (trans-OH-taurine) which was first generated by stereoselective reduction of the double bond and the carbonyl function of the CS-670 molecule. The taurine conjugate was excreted in urine as the main metabolite, regardless of the optical configuration of CS-670 administered [2R)-enantiomer: 47.2% of the dose, (2S)-enantiomer: 70.9% of the dose]. The trans-OH-taurine was hydrolyzed by refluxing it in 6 N HCl without racemization. The released trans-OH was derivatized to diastereoamides with (+)-(R)-1-(1-naphthyl)ethylamine to examine the stereochemical properties of the 2-arylpropionic acid side chain. It was found that the configuration of the 2-carbon of the trans-OH-taurine was almost entirely (S). As the CoA thioesters are obligate intermediates for amino acid conjugation, the results suggest that the (2S)-enantiomer of the trans-OH metabolite serves as a substrate for canine acyl CoA ligase (EC 6.2.1.3) as well as the (2R)-enantiomer, but only the CoA thioester with a (2S)-configuration is a substrate for taurine N-acyl transferase. It is interesting to note that these results are not consistent with the chiral inversion mechanism by which the (2R)-enantiomers of profen NSAIDs are stereospecifically converted to CoA thioester intermediates.

Quantification of the Na+/K(+)-pump in solubilized tissue by the ouabain binding method coupled with high-performance gel chromatography.

Membrane-bound Na+/K(+)-ATPase purified from dog kidney outer medulla was solubilized with octaethylene glycol n-dodecyl ether (C12E8) and incubated with [3H]ouabain in the presence of NaCl. ATP and MgCl2 for 10 min at 0 degrees C. The resulting enzyme was separated, by high-performance gel chromatography executed at 0.2 degrees C. Mainly into its (alpha beta)2-diprotomer and alpha beta-protomer, which both bound stoichiometrically to [3H]ouabain. The amounts of ouabain that bound to the tissue itself and its microsomes could be estimated in the same way, as [3H]ouabain was found to bind only to the diprotomer and protomer they possessed. The amounts of ouabain that bound to them in the solubilized state were at least 5-times higher than those that did so when they were non-solubilized, suggesting that the surfactant rendered the enzyme accessible to ouabain. When the solubilized tissue (138 mg ml-1 wet tissue) was reacted with ouabain in the presence of 0.1 M NaCl and 4.8 mM MgCl2 for 10 min at 0 degrees C, maximal ouabain binding was attained in the presence of 18.3 microM [3H]ouabain, 1.2 mM ATP and 3 to 5 mg ml-1 C12E8, which was common to the outer medulla and human colon cancer cells. The present method enabled the pump number in protein and tissue samples in the range 7.2 x 10(-9) (purified pump) to 1.5 x 10(-12) (cancer tissue) mol/mg protein to be estimated within 2 h.

Quantitative determination of diol metabolites of CS-670, a new antiinflammatory agent, by capillary column gas chromatography-mass spectrometry.

CS-670(I), being developed as a non-steroidal anti-inflammatory agent, is a racemic prodrug. It has been found to be readily metabolized to active metabolites: trans and unsaturated mono-ols (trans-OH, unsaturated-OH). We report here a method for the quantitative determination of the eight diol stereoisomers excreted in urine after administration I. The diols were well separated and quantitated using capillary column GC-MS after a rather simple derivatization with diazomethane-trifluoroacetic anhydride. Sex differences in rats and species differences between rats and mice were observed in the metabolism of I: the trans-diols originating from trans-OH were predominantly excreted in male and female rat urine but the excretion rate was greater in the male rats; the cis-diols originating from cis mono-ol (cis-OH) were the major urinary metabolites in mice. The hydroxy groups were mainly introduced at the respective equatorial hydrogen atoms at the 4'-carbon of trans-OH and the 5'-carbon of cis-OH. The 4'- and 5'-hydroxy groups in the diols were in the cis conformation with respect to the original 2'-hydroxy group. As approximately 9% of the trans-diols were excreted in urine after administration of cis-OH to rats, the chiral inversion from cis-OH to trans-OH was suggested to occur through the saturated ketone intermediate.

Immunocytochemical localization of estrogen and progesterone receptors (ER & PgR) in breast cancer.

The localization of estrogen receptors (ER) and progesterone receptors (PgR) in human breast cancer was examined light and electron microscopically by using specific monoclonal antibodies. Among the breast cancer patients, ER-positive (ER[+]) cases were shown by enzyme immunoassay (EIA) to account for 58.9% (63/107), and 63.5% of these cases were women in the postmenopausal state. The PgR-positive (PgR[+]) rate was 50.5% (54/107), with 44.4% of these positive cases being postmenopausal women. The rates of ER(+) and PgR(+), ER negative (ER[-]) and PgR negative (PgR[-]), and ER(+) and PgR(-) cases were 43.9% (47/107), 34.6% (37/107) and 15.0% (16/107), respectively. Although ER(-) and PgR(+) cases were in few number, they were found (6.5%, 7/107). These results correlated well with those obtained by the immunocytochemical method. In either case, i.e anti-ER or anti-PgR reaction, positive nuclei and negative nuclei were found intermingled with each other in a given visual field. In electron microscopy, both anti-ER and anti-PgR antibodies bound to sites in the euchromatin area of the nucleus.

Isolation and characterization of CAJ1, a novel yeast homolog of dnaJ.

A novel member of the Escherichia coli dnaJ family, designated CAJ1, was isolated from a yeast expression library using antiserum against a yeast calmodulin-binding fraction. Although CAJ1 contains neither a Gly-rich region nor a Cys-rich repeat, as are found in other DnaJ relatives, it contains a leucine zipper-like motif.

Detection of the FK506-FKBP-calcineurin complex by a simple binding assay.

[3H]FK506-FK506-binding protein (FKBP) complex does not bind polyethyleneimine-treated glass fiber filters, on the other hand, it binds the filter when it makes a complex with calcineurin. This enabled us to develop a simple binding assay to detect the FK506-FKBP-calcineurin complex in the mixture and to estimate the amount of FKBP or calcineurin which can form the complex in the soluble extracts from rat tissues. The molar concentration of FKBP of every rat tissue is much higher than that of calcineurin, and FKBP/calcineurin molar ratio varies from 13 to 33 in various brain regions, and from 42 to 343 in peripheral tissues. These results suggest that most of the FKBP molecules have physiological roles which are not related to calcineurin, in spite of their potential ability to form the complex with FK506 and calcineurin.

Stereoselective determination of the active metabolites of a new anti-inflammatory agent (CS-670) in human and rat plasma using antibody-mediated extraction and high-performance liquid chromatography.

The main metabolites of (+-)-2-[4-(2-oxocyclohexylidenemethyl)phenyl]propionic acid (CS-670), a new pro-drug anti-inflammatory agent of the 2-arylpropionic acid type, have one or two chiral centres arising from reduction of the oxocyclohexylidene moiety in addition to an original chiral centre in the propionic acid moiety. To determine these metabolites stereoselectively, antibody-mediated extraction was investigated as a stereoselective clean-up method prior to chiral HPLC. Immunoglobulin G, which recognizes each stereoisomeric cyclohexanol moiety, was coupled to cyanogen bromide-activated Sepharose 4B to prepare re-usable immobilized antibody, and its specificity was improved by examination of a washing process after charging of samples. Plasma extracted with the immobilized antibody column was derivatized with a chiral reagent to separate the enantiomers of the propionic acid moiety by HPLC. This newly developed analytical method clarified the stereoselective biotransformation of the pro-drug to pharmacologically active forms in humans and rats related to reduction of the oxocyclohexylidene moiety and chiral inversion in the propionic acid moiety.

Appearance of adherent cells suppressive to erythropoiesis during an early stage of Plasmodium berghei infection in mice.

We examined the effect of adherent cells from bone marrow or spleen of mice infected with Plasmodium berghei on dyserythropoiesis. Significant reduction in number of erythroid progenitors (erythroid colony-forming units: CFU-E and erythroid burst-forming units: BFU-E) was observed in bone marrow as early as 1 day after P. berghei infection. When adherent cells were removed from bone marrow or spleen cells of infected mice, the number of CFU-E and BFU-E was clearly increased. Furthermore, addition of adherent cells from infected mice to nonadherent cells from normal mice inhibited erythroid colony formation significantly in a dose-dependent manner. These results suggest that the adherent cells obtained from bone marrow or spleen of mice in the early stage of P. berghei-infection have a suppressive effect on erythropoiesis.