The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

Fluorometric determination of plasma adenosine concentrations using high-performance liquid chromatography

Methods are described for the fluorometric determination of plasma adenosine concentrations, using HPLC. Plasma obtained from blood of dogs treated with erythro-(2-hydroxy-3-nonyl)adenine hydrochloride and dipyridamole was deproteinized with perchloric acid and the neutralized sample was put sequentially onto a SepPak C18 and boronic acid affinity column. Subsequently, adenosine in the final elution was converted to 1,N6-ethenoadenosine and was quantitated by HPLC with a fluorescence detector. The percentage recovery of adenosine added to the deproteinized plasma was nearly 100%. In the adenosine deaminase treated plasma, the increase in adenosine concentration of even 4 nM can be accurately determined. The control renal venous plasma concentrations of adenosine in anesthetized dogs were 19.9 +/- 1.9 nM, a significantly higher value than the corresponding arterial concentrations (12.7 +/- 1.1 nM), thereby suggesting the renal release of adenosine. This release was markedly enhanced following the removal of the renal arterial occlusion. Thus, taken together with the in vivo results, the present method is sensitive, hence most useful for the determination of plasma adenosine concentrations.     

Effect of bradykinin on airway neural responses in vitro.

We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.     

Inhibitory NANC nerves in human tracheal smooth muscle: a quest for the neurotransmitter.

Inhibitory nonadrenergic noncholinergic (i-NANC) nerves are the only neural bronchodilator pathway in human airways. Possible candidates for the neurotransmitter include vasoactive intestinal peptide (VIP) and nitric oxide (NO) and purines such as ATP. We have investigated the potential role of these neurotransmitters. Phosphoramidon (10(-5) M) significantly potentiated relaxations to low doses of VIP with no effect on i-NANC responses. Relaxations induced by VIp were abolished with alpha-chymotrypsin (2 U/ml), but i-NANC responses were unaffected. Reactive blue 2 had no effect on i-NANC neural responses, indicating that endogenous ATP was not involved. The NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10(-4) M) produced a concentration-dependent inhibition of the i-NANC response, producing almost complete inhibition at every frequency studied (0.5-40 Hz), whereas L-NG-monomethyl arginine was effective only at low stimulation frequencies. The inhibitory effect of L-NAME was partially reversed by L- but not D-arginine, and D-NAME was without effect. These results suggest that in human tracheal segments the neural bronchodilator response is mediated by NO, and there is no functional evidence for implicating VIP in this response.     

Estimation of the degree of self-fertilization in Porphyra yezoensis (Bangiales,Rhodophyta)

Crosses between genotypically distinct thalli of the monoecious species Porphyra yezoensis were carried out using immature thallus fragments from green- and red-type color mutants and also wild-type thalli. As the genes governing the mutants are monogenic, recessive to the wild-type, and belong to the same linkage group, the degree of self-fertilization could be estimated based on the pigmentation of the resultant diploid conchocelis. The degree of self-fertilization in the cross between the green-type and the wild-type was 48.5–55.0%, and in the cross between the red-type and the wild-type was 45.1–56.5%. In the cross between the green- and red-type mutants, the degree of self-fertilization was 46.0–54.5% when the green-type was the female parent, and was 44.8–55.6% when the red-type was the female parent.     

The effects of planktivorous fishes on the plankton community in a eutrophic lake

Stocking silver carp, a phytoplankton feeder, and bighead carp, an omnivorous plankton feeder, into an eutrophic lake at high densities caused a dramatic change in the lake ecosystem.Microcystis, which had been dominant in summer and a main food of the fishes decreased markedly, and green algae smaller than 10 μm then became dominant. Consequently, chlorophyll-a per unit area decreased slightly, while the rate of production was higher than that in the previous years. As the total density of the fishes increased (from 0.09 to 0.11 fish m⁻²), the growth of silver carp was retarded, while that of bighead carp increased.Microcystis, was unable to become dominant due to increased grazing pressure by the fishes, and small green algae became dominant. The lake conditions thus became more favourable for zooplankters which selectively consumed small green algae, and accordingly, the production of zooplankton rose. Bighead carp consumed more animal food, which they assimilate at a higher rate than plant food, and grew better in spite of the fact that the fish density increased. The feeding rate of silver carp was greatly reduced because the green algae were too small to ingest, and the fish therefore grew poorly. Results of a computer simulation of a model consisting of five compartments, representing the blue-green algae, green algae, zooplankton, silver and bighead carp, support the food-web change observed in the lake.     

Localization of aldosterone-producing adenoma: venous sampling in primary aldosteronism

Adrenal venous sampling of blood was performed for nine patients with aldosterone-producing adenoma (APA). Measurement of adrenal venous aldosterone is useful for localization of APA but difficult, because catheterization of the right adrenal vein is not easy, and the blood is diluted by nonadrenal flow. To solve these problems, levels of aldosterone (A; ng/dl) and cortisol (C; micrograms/dl) were measured in samples from the left adrenal vein (LAV) and the inferior vena cava (IVC), and the LAV A/C and (LAV A/C)/(IVC A/C) ratios were calculated. These ratios were also obtained for 16 patients with essential hypertension. The adenoma could be localized in three of the nine cases by the measurement of aldosterone alone, but the use of a LAV A/C ratio greater than 5 x 10(-3) and a (LAV A/C)/(IVA A/C) ratio less than 1.0 as criteria separated the patients into those with a left APA, right APA, or essential hypertension. Consequently, adrenal venous sampling and the calculation of these ratios enables preoperative localization of APA with more accuracy, especially when the tumor is small or the result of CT and adrenal scintigraphy is not consistent.     

Seroepizootiological survey on bluetongue virus infection in cattle in Japan

Bovine sera collected in various parts of Japan were subjected to seroepizootiological tests with bluetongue virus type 1 (BTV1), type 12 (BTV12), and type 20 (BTV20). All these viruses have been widely disseminated among cattle in the southern part of Japan in 1974. Relatively high incidences of neutralizing (NT) antibody against the three viruses were shown among cattle in the Kyushu district, including Okinawa Prefecture, or the southern part of Japan, but extremely low or incidences in Hokkaido, or the northern part of Japan. The incidence of reactors was higher in old animals. Cattle in Okinawa Prefecture showed a high rate of seroconversion for all the viruses during the summer of 1979. None of the animals seroconverted, however, manifested any sign of disease. Seroepizootiological investigation made it clear that BTV1, BTV12 and BTV20 had existed in Japan and that the epizootic of bluetongue virus infection started during a period from summer through early autumn.     

Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.