Cytochemical and electron microscopic demonstration of organelle translocation and the inhibition of such translocation by colchicine during the mitosis of rat hepatocytes

Hepatocyte lysosomes, mitochondria, and peroxisomes show a dramatic translocation during mitosis induced by partial hepatectomy. During prophase, all three organelles move to the perinuclear cytoplasm. In metaphase, they become concentrated in the polar regions. During telophase, these organelles form clusters in the juxtanuclear regions. This organelle translocation is inhibited by the administration of a low concentration of colchicine, suggesting an involvement of microtubules in their movement.     

A specific heterotypic cell adhesion activity between members of carcinoembryonic antigen family, W272 and NCA, is mediated by N-domains

The Ca(2+)-independent homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, W272 (CGM6), whose cDNA has recently been isolated from libraries of human peripheral leukocytes of apparently normal subjects (Arakawa, F., Kuroki, Mo., Misumi, Y., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1063-1071) and spleen of chronic myelogenous leukemia patients (Berling, B., Kolbinger, F., Grunert, F., Thompson, J. A., Brombacher, F., Buchegger, F., von Kleist, S., and Zimmermann, W. (1990) Cancer Res. 50, 6534-6539) has been examined. Chinese hamster ovary cells transfected with the cDNA for W272, CEA, nonspecific cross-reacting antigen (NCA), and various antigens containing chimeric N-domain have been used. The W272 producers did not show homotypic binding at all but bound only to the cells expressing NCA and a chimeric CEA whose N-domain is substituted by that of NCA, indicating the major contribution of N-domain of NCA in the specific binding. The importance of the N-terminal region of NCA N-domain for the W272-NCA binding has been shown by detailed analysis using COS-1 cells producing various NCA whose N-domain are chimera of that of NCA and CEA. The strict heterotypic nature of the W272-NCA adhesion strongly suggests that the cell adhesion activities exhibited by CEA family members are not the fortuitous activity but the specific one which have some important physiological roles.     

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.     

High oxygen transfer rate in a new aeration system using hollow fiber membrane

A new type of bubble aeration column called a hollow fiber membrane (HFM) aeration column was proposed, which was featured in the use of hollow fiber membranes and gave a high bubble density in the column. The value of k(L)a was increased by modifying the membrane surface for making the pore size smaller. The Sauter mean diameter of bubbles (D(vs)) was 2.0 +/- 0.1 mm in the range of the superficial gas velocity from 0.02 m s(-1) to 0.065 m s(-1), while that obtained for the bubbles near the membrane was 811 mum at the superficial gas velocity of 4.0 x 10(-4) m s(-1). The difference was ascribed to the effect of coalescence of bubbles. The value of K(L)a increased in proportion to the superficial gas velocity up to 0.02 m s(-1), and was almost constant above 0.03 m s(-1). The maximum value of k(L)a, 2.5 s(-1), was higher than those of the other aeration columns reported previously. The pneumatic power consumption per unit liquid volume (P(v)) for obtaining the same k(L)a was the smallest in the HFM aeration columns. P(v), for obtaining the same interfacial area of bubbles per liquid volume, was also lower than those for other types of aeration columns. It was suggested from the measurement of bubble diameter that the larger interfacial area generated in the HFM aeration column ascribes to the larger gas holdup than the smaller D(vs). (c) 1992 John Wiley & Sons, Inc.     

Life cycle of Gnathostoma nipponicum Yamaguti, 1941.

The life cycle of Gnathostoma nipponicum was examined by field survey and by experimental infection of animals with the larvae. Naturally infected larval G. nipponicum were found in loaches, catfish, and snakes. Experimentally, loaches, killifishes, frogs, salamanders, mice, and rats were successfully infected with the early third-stage larvae of G. nipponicum obtained from copepods (the first intermediate host), whereas snakes, quails, and weasels were not. Frogs, snakes, quails, and rats were experimentally infected with the advanced third-stage larvae (AdL3) from loaches. These results reveal that some species of fishes, amphibians and mammals can act as the second intermediate host and that some species of reptiles, birds and mammals can act as a paratenic host. The life cycle was completed in weasels, the definitive host, which were infected with AdL3 from loaches and started to evacuate eggs of G. nipponicun in faeces on days 65-90 postinfection.     

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.     

Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test.

The in vitro micronucleus (MN) test was carried out simultaneously with the conventional chromosomal aberration (CA) test on 11 clastogenic chemicals or spindle poisons with different modes of action using a Chinese hamster cell line (CHL). The method of slide preparation for the MN test was the same as that for the conventional metaphase analysis, except that 1% acetic acid in methanol was used as the cell suspension medium for air-drying (to preserve the cytoplasm around the nucleus). All chemicals tested induced micronuclei reproducibly and dose-dependently in good agreement with the results of metaphase analysis (r = 0.99). Since the MN test methodology is simple and the observation of MN is less subjective than that of CA, we conclude that the in vitro MN test would be a good alternative to the conventional CA test for screening the genotoxicity of chemicals.     

Micronucleated reticulocyte induction by ethylating agents in mice.

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.     

Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene.

Micronucleus assays using mouse peripheral blood stained vitally on acridine orange (AO)-coated slides were evaluated at two laboratories with 7,12-dimethylbenz[a]anthracene (DMBA) and compared with the standard bone marrow assay. DMBA was administered by single intraperitoneal injection to CD-1 mice at doses ranging from 5 to 80 mg/kg, then 5 microliters of peripheral blood was sampled from a tail vein at 24, 48, 72, 96, and 120 h after treatment. Similar incidences of micronucleated young erythrocytes were observed in peripheral blood reticulocytes and bone marrow polychromatic erythrocytes. The dose response of micronucleated reticulocytes was delayed compared to that of micronucleated polychromatic erythrocytes. The dose-response curves after treatment with DMBA differed depending on the sampling times, which revealed the difficulty of obtaining accurate dose-response relations in the micronucleus assay. The present result demonstrated that the simple and rapid AO supravital staining method is a valuable and easier method for obtaining dose- and time-response data for quantification of micronucleus induction by chemicals.