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.

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.

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.

Hepatic eosinophilopoiesis from multipotent hemopoietic stem cells in Toxocara canis-infected mice.

Extramedullary hemopoiesis, recognized as hemopoietic foci, increased in the livers of Toxocara canis-infected mice. At the peak of the response (day-13 after infection), the majority of hepatic hemopoietic foci were of the eosinophil lineage. Hepatic nonparenchymal cells prepared from T. canis-infected mice on day 13 contained large numbers of hemopoietic stem cells, more than half of which were cycling. When W/Wv mice, which are genetically deficient in multipotent hemopoietic stem cells, were infected with T. canis, hepatic hemopoietic foci were rare throughout the course of infection. This impaired response of W/Wv mice was restored by bone marrow grafting from normal +/+ littermates. These results indicate that, in response to the increased demand, eosinophils are generated in the liver by the differentiation from multipotent stem cells, not only from the committed precursors.

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.

Inhibition of prostaglandin production in the inflammatory tissue by loxoprofen-Na, an anti-inflammatory prodrug.

The effect of loxoprofen-Na, a novel non-steroidal anti-inflammatory drug with a prodrug property, on prostaglandin (PG) levels in the inflammatory tissue was investigated with a carrageenin-induced pleurisy model in rats. The intrapleural injection of carrageenin caused a marked increase in the levels of PGE2 and 6-keto-PGF1 alpha in the pleural exudate up to 3 hr after the injection. When [14C]PGE2 was injected into the cavity 2 hr after the carrageenin injection, the PG rapidly disappeared from the cavity (T 1/2 = 5 min). Thus, the PG level determined in the inflammatory exudate represents PG produced in the inflammatory tissue. Loxoprofen-Na, administered orally 2 hr after the carrageenin injection, dose-dependently inhibited the increase in the levels of PGs in the exudate 1 hr after administration (ID50 = 0.07 mg/kg for PGE2 and 0.10 mg/kg for 6-keto-PGF1 alpha). Indomethacin also inhibited PG production, but was less effective (ID50 = 0.24 mg/kg for PGE2 and 0.47 mg/kg for 6-keto-PGF1 alpha). Similar results were obtained 3 hr after the administration of these drugs (ID50 of PGE2 production = 0.14 mg/kg for loxoprofen-Na and 0.28 mg/kg for indomethacin). The time-course analysis of the effect of loxoprofen-Na showed that this drug had more immediate and stronger inhibitory activity than indomethacin. The relative potencies of suppression of protein leakage and leukocyte infiltration correlated well with the inhibition of PG production, but higher doses were needed for an obvious anti-inflammatory effect. The active metabolite (SRS trans-OH) of loxoprofen-Na determined in the inflammatory exudate 1 hr after oral administration of 0.2 and 2 mg/kg of loxoprofen-Na was 0.05 and 0.25 micrograms/mL, respectively. The concentration was sufficient to suppress PG production in the exudate, because the IC50 of the SRS trans-OH for PG production in vitro with leukocytes was 0.02 microgram/mL (0.01 microM). The potency of the SRS trans-OH metabolite to inhibit PGE2 production in leukocytes was about 20 times stronger than that of the parent compound and 3 times stronger than that of indomethacin.

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.

A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria.

The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17x-infected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen: CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.

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.

Lack of correlation between lysosomal enzyme activity and ascorbic acid content in brain of lead-treated animals.

The administration of lead acetate increased acid phosphatase activities in the rat brain and four regions (cerebral cortex, diencephalon plus mesencephalon, pons plus medulla, and cerebellum) of the guinea pig brain, whereas ascorbic acid content in the brain of these animals remained unchanged following lead administration. Acid phosphatase activity also showed an increase in scorbutic guinea pig brain. Lipid peroxidation facilitated by ascorbic acid in the cerebral lysosome fraction of rat was not affected by lead. These results suggest that ascorbic acid is not directly concerned with the alteration in acid phosphatase activity induced by lead treatment.

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.

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.

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.

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.

[Localization of glycylproline dipeptidyl aminopeptidase in the liver tissue and possible mechanism of its release into blood flow].

We examined the localization of glycylproline dipeptidyl aminopeptidase (GPDAP) in the liver tissue and the mechanism of its release into blood flow. An immunohistochemical study using rabbit polyclonal antibody against the purified GPDAP from human liver obtained at autopsy was performed in liver biopsy samples with the peroxidase-antiperoxidase stain technique. GPDAP staining was detected on the liver cell membrane around bile canaliculi. After treatment with deoxycholic acid (DOC) or Triton X-100, this enzyme disappeared. GPDAP was solubilized rapidly from microsome of human liver by treatment with either DOC or Triton X-100. DOC or Triton X-100 solubilized GPDAP coincided with the isozyme that was the specific isozyme in sera of patients with acute hepatitis or obstructive jaundice. These results suggest that GPDAP localizes on the liver cell membrane around bile canaliculi and that this enzyme is released by the detergent action of bile acids.

[Management of cesarean section under replacement therapy with factor VIII concentrates in a pregnant case with congenital combined deficiency of factor V and factor VIII].

The congenital combined deficiency of Factor V and Factor VIII, a rare bleeding disorder, was identified in a 25-year-old woman. She was admitted to our hospital with a complaint of genital bleeding. Her prothrombin time and activated partial thromboplastin time were prolonged. She had low levels of Factor V coagulant activity (F. V:C) 14%, and Factor VIII coagulant activity (F. VIII:C), 12%, and normal levels of von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (Rcof) and Protein C antigen. Her Protein C inhibitor level was slightly low. Her Rcof, vWF:Ag and F. VIII:C were elevated following administration of 1-deamino-8-D-arginine-vasopressin (DDAVP), but her F. V:C remained unchanged. Four years later, her F. VIII:C rose to 70% during the course of her pregnancy, but her F. V:C value remained low. It was expected that the vaginal delivery would be possible at the termination of pregnancy. Premature rupture of the membranes and an anomaly of rotation appeared in the course of delivery, however, and cesarean section was accomplished without excess bleeding under replacement therapy with Factor VIII concentrates. These findings suggested that DDAVP and Factor VIII concentrates were useful for management of her delivery. However the mechanisms of the rise of plasma F. VIII:C during pregnancy in a case with congenital combined deficiency of Factor V and Factor VIII are unclear.

[B-chronic lymphocytic leukemia/prolymphocytic leukemia (CLL/PL)--a case report].

A 48-year-old male was admitted to our hospital on April 20, 1989 because of general fatigue and abdominal fullness. Physical examination showed hepatomegaly, massive splenomegaly, and systemic lymphadenopathy. Hematological findings revealed WBC 73,000/microliters, RBC 289 x 10(4)/microliters, Hb 8.0g/dl, and platelet 9.1 x 10(4)/microliters. WBC differential count demonstrated a mixture of 63% matured small lymphocytes and 32% prolymphocytoid cells. Bone marrow aspiration was unsuccessful with a dry tap. Surface marker analysis of peripheral blood lymphoid cells disclosed that they were positive for anti-HLA-DR, CD 5, CD 19, CD 20, CD 21, CD 25, Sm-IgM, Sm-IgD, and Sm-K. He was diagnosed as B-CLL/PL, and treated with VEPA with partial remission. CLL/PL which was advocated by Melo in 1986 is regarded as a distinct clinical entity intermediate between CLL and PLL in clinical and laboratory features. Our case is interesting with regard to good response to combination chemotherapy, though most cases of CLL/PL have a resistance to standard chemotherapy.

[Analysis of the denture dynamics in R.P.D.'s. 2. Influence of retainers on the dynamics of free-end-saddle].

The ordinary denture movement during function is defined as the "Denture dynamics" in R.P.D.'s. by K. H. Körber (1975). The Author has already reported the evaluating method of the denture dynamics objectively by two ways: The displacement sensors of magnetoresistors detected the vertical and horizontal movements of denture saddle in free end situation. Tiny accerelometer also detected the accerelation during the movements. In this present study, the correlations among the retainers used, the extent of the denture dynamics in displacement and accerelation, the occluding force and the abutment tooth mobility induced by the denture support were correlatively evaluated in two patients with class I free end R.P.D.'s. with interchangeable retainers by above methods. All the data were collected simultaneously by the electronic measuring and recording apparatus. Following results were obtained: 1. The denture dynamics depended on the selection of retainers examined. The extent of the dynamics reduced from the denture with the wrought wire clasps, the denture with the cast clasps, then to the denture with cone crown telescopes in sequence. 2. The occluding force increased just as the changes in sequence of the denture dynamics. 3. The abutment tooth moved to the distal in each case. The highest value of the mobility was found in the denture with the cast clasps, then in the denture with the wrought wire clasps. The least value of the tooth mobility was found in the denture with the cone crown telescopes. These results will strongly support the concept of the free end saddle R.P.D.'s anchored and supported rigidly by the abutments and the residual ridges in clinics.