The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

(-) deprenyl induces activities of both superoxide dismutase and catalase but not of glutathione peroxidase in the striatum of young male rats

Daily s.c. injection of (-)deprenyl (2.0 mg/kg/day) for three weeks in young male rats caused a threefold increase in superoxide dismutase (SOD) activity in the striatum of the brain compared with the value in saline-injected control rats. Furthermore, the activity of catalase (but not of glutathione peroxidase) was also increased significantly by deprenyl treatment. The results confirmed the previous findings of Knoll on SOD activity and furthermore provided evidence that the activity of catalase is also significantly induced by the drug, which was not found in the previous study.     

Molecular properties of ornithine decarboxylase from mouse kidney: detailed comparison with those of the enzyme from rat liver

Ornithine decarboxylase (ODC) was purified about 2,000-fold from the kidney of androgen-treated mice and its molecular properties were examined and compared with those of the enzyme from rat liver. The purified enzyme showed two protein staining bands on SDS-polyacrylamide gel electrophoresis, corresponding to Mr of about 54,000 and 52,000. The apparent Mr of the enzyme determined by gel filtration was 57,000 in the presence of 0.25 M NaCl and 110,000 in its absence. The apparent Km value for L-ornithine was about 0.1 mM in the absence of NaCl and 0.7 mM in the presence of 0.25 M NaCl. Thus, salts appeared to cause subunit dissociation and also an increase in the Km value for the substrate. Putrescine and D-ornithine acted as inhibitors competing with the substrate. Antizyme from the rat liver inhibited the activities of the mouse enzyme and the rat enzyme similarly. The mouse and the rat enzymes exhibited a very similar immunological cross-reactivity to rabbit antibody raised against the mouse enzyme but, when the antibody directed against the rat enzyme was used, the cross-reactivity of the rat enzyme was higher than that of the mouse enzyme. Thus, the molecular properties of mouse ODC were very similar to those of the rat enzyme.     

The order of hepatic cytotoxicity of bile salts in vitro does not agree with that examined in vivo in rats

Using an enzyme release from isolated rat hepatocytes incubated with a bile salt as a marker, the cytotoxic order of bile salts was found to be taurochenodeoxycholate, glycochenodeoxycholate greater than tauroursodeoxycholate, glycoursodeoxycholate, cholate greater than taurocholate, glycocholate. Thus, the cytotoxicity of conjugates of ursodeoxycholate was greater than that of conjugates of cholate. However, these data do not agree with the order of cytotoxicity of these bile salts previously studied in vivo by the authors which demonstrated the least cytotoxic nature of conjugates of ursodeoxycholate.     

Mutations associated with floral organ number in rice

How floral organ number is specified is an interesting subject and has been intensively studied in Arabidopsis thaliana. In rice (Oryza sativa L.), mutations associated with floral organ number have been identified. In three mutants of rice, floral organ number 1 (fon1) and the two alleles, floral organ number 2-1 (fon2-1) and floral organ number 2-2 (fon2-2), the floral organs were increased in number centripetally. Lodicules, homologous to petals, were rarely affected, and stamens were frequently increased from six to seven or eight. Of all the floral organs the number of pistils was the most frequently increased. Among the mutants, fon1 showed a different spectrum of organ number from fon2 -1 and fon2 -2. Lodicules were the most frequently affected in fon1, but pistils of more than half of fon1 flowers were unaffected; in contrast, the pistils of most flowers were increased in fon2 -1 and fon2-2. Homeotic conversion of organ identity was also detected at a low frequency in ectopically formed lodicules and stamens. Lodicules and stamens were partially converted into anthers and stigmas, respectively. Concomitant with the increased number of floral organs, each mutant had an enlarged apical meristem. Although meristem size was comparable among the three mutants and wild type in the early phase of flower development, a significant difference became apparent after the lemma primordium had differentiated. In these mutants, the size of the shoot apical meristem in the embryo and in the vegetative phase was not affected, and no phenotypic abnormalities were detected. These results do not coincide with those for Arabidopsis in which clavatal affects the sizes of both shoot and floral meristems, leading to abnormal phyllotaxis, inflorescence fasciation and increased floral organs. Accordingly, it is considered that FON1 and FON2 function exclusively in the regulation of the floral meristem, not of the vegetative meristem.Abbreviation DIC differential interference contrast This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.     

Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit-shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10^?8 joules per μm² was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time-lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half-bivalent with the partner half-bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.     

Photomicrobial electrode for selective determination of sulphide

Summary A photomicrobial electrode, which uses the photosynthetic bacteria Chromatium sp. in conjunction with a hydrogen electrode, was developed for the determination of sulphide. The response time of the photomicrobial electrode was 5–10 min. A linear relationship was obtained between the current of the electrode and the sodium sulphide concentration below 3.5 mM. The minimum detectable concentration of sodium sulphide was 0.4 mM. Selectivity of the sensor is satisfactory. A good agreement was obtained between the photomicrobial electrode and the ethylene blue method (correlation coefficient: 0.90).     

Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: a flow cytometric analysis

Dormant lymphocytes are known to show little LDL receptor (LDL-R) activities. The present study was designed to determine whether or not LDL-R activities of lymphocytes from normal subjects were high enough to be measured by flow cytometry after the cells had been stimulated with recombinant interleukin-2 (IL-2) and anti-CD3 monoclonal antibody (mAb). IL-2 or anti-CD3 mAb individually provokes proliferation of lymphocytes in a serum-free medium. Proliferation rate was accelerated when the two reagents were used in combination. Stimulated cells cultured for 5 days expressed more than 85% CD3 positive, less than 0.5% CD14 positive, and less than 1.5% CD20 positive. The LDL-R activities of the cells were examined by the uptake of a fluorescence probe, DiI-labeled LDL (DiI-LDL) and analyzed by flow cytometry. Stimulated cells showed increased uptake of DiI-LDL and 84 +/- 9% were positive, whereas only 3.0 +/- 2.5% of the cells without stimulation were positive (P less than 0.001). Under the same conditions stimulated lymphocytes from a homozygous familial hypercholesterolemia (FH) patient showed little LDL-R activities; 14% of the cells were positive. Displacement assays reveal that the uptake of LDL by these cells is occurring by way of its specific pathway. These data imply the lymphocytes stimulated with the reagents used in the study might be used for detecting defects in LDL-R, perhaps defects in other genomic systems as well.