Kosaku Izutsu

The effect of K + on the membrane potential in HeLa cells.

Abstract 1. 1. The membrane potential in HeLa cells was determined with the use of a microelectrode under visual control with a phase contrast microscope at a high magnification. 2. 2. The mean value of the potential for 158 cells in culture media at 37 °C was −48.1 ± 0.95 mV (SE), interior negative, while the mean for 297 cells in the control Dulbeccos phosphate buffer solution was −48.2 ± 0.68 mV. 3. 3. When chloride in the medium is partially replaced with sulphate at a fixed external K + concentration, [K + ] 0 , the membrane potential was scarcely affected, whereas marked changes in the potential were observed with varying [K + ] 0 under a constant [Cl − ] 0 . 4. 4. Such a change in the membrane potential caused by varying [K + ] 0 obeys Nernsts equation in the range of [K + ] 0 higher than 20 mM, but deviates largely from the equation at lower K + concentrations. The potential increases with decreasing [K + ] 0 . 5. 5. Using Wickson-Ginzburg and Solomons data ( J. Gen. Physiol. , 46 (1963) 1303) on the ionic composition of HeLa cells, the value of P Na / P K in the range of [K + ] 0 less than 20 mM was estimated from Goldmans equation ( J. Gen. Physiol. , 27 (1943) 37) under the assumption that Cl − never contributes to the membrane potential. The ratio of the permeability constants markedly increases with decreasing [K + ] 0 . Such an increase in the ratio P Na / P K , was discussed from the viewpoint of permeability of non-excitable cell membranes.

Instability of pleomorphic tubulin paracrystals artificially induced by Vinca alkaloids in tissue-cultured cells.

The processes of tubulin paracrystal induction in Chinese hamster ovary cells treated with a Vinca alkaloid, ie, vinblastine or vincristine, and treated simultaneously with one of the Vinca alkaloids and colcemid or colchicine were followed by four different microscopic techniques, in particular by tubulin‐immunofluorescence. Vinca alkaloid alone, in lower concentrations, induced basically tactoid or needle‐shaped (N‐shaped) paracrystals. However, the formation of crystalloid was greatly enhanced by increasing the concentration of Vinca alkaloid. Square or barrel‐shaped (S‐shaped) and hexagonal paracrystals were also commonly induced by simultaneous treatment with a Vinca alkaloid and colcemid or colchicine. Large rectangular paracrystals often displayed fibrillar or lamellar fine structures which ran perpendicular to the long axis but tended to cleave into fragments by spontaneous splitting. Electron micrographs revealed the fine structure of crystalloids to be aggregates of numerous filaments. The growth of paracrystals, particularly N‐shaped crystals, was markedly inhibited when cells were exposed to drug(s) at a low temperature (4°C). We confirmed that both N‐ and S‐shaped paracrystals dissociated rapidly after the culture medium was replaced with fresh, drug‐free medium. Glutaraldehyde‐fixed paracrystals treated with RNase solution were stained with acridine orange, showing a weak orange color. Possible factors involved in the assembly and disassembly of tubulin paracrystals are discussed.

Studies on endoreduplication III. Endooreduplication induced by mitomycin C in PHA-stimulated tonsillar lymphocyte cultures

The activity of mitomycin C (MMC) to induce endoreduplication was examined in PHA-stimulated tonsillar lymphocyte cultures. Cellular kinetics of both diploid and endoreduplicated cells was studied by the combined techniques of thymidine and BrdU incorporation. The kinetic data showed that the increase of endoreduplicated cells induced by MMC correlated with the increase of the second-generation diploid cells. Also, it showed that the majority of endoreduplicated cells found at the time when the maximal number of endoreduplicated cells was obtained had incorporated BrdU only once into their diplochromosomes. This suggests that MMC-induced endoreduplicated cells originate from MMC-induced G2-arrested cells.

Lymphocyte calmodulin and its participation in the stimulation of T lymphocytes by mitogenic lectins

Summary— Calmodulin was purified from human tonsillar lymphocytes utilizing calcium‐dependent binding of calmodulin to fluphenazine‐Sepharose. The molecular weight and phosphodiesterase activation of the lymphocyte calmodulin were very similar to those of purified bovine brain calmodulin. Trifluoperazine (TFP), a calmodulin inhibitor, suppressed lymphocyte stimulation as assessed by 3H‐thymidine incorporation into DNA of lectin‐stimulated lymphocytes. TFP had no effect on the early 45Ca2+ uptake induced by mitogenic lectins, although this latter was inhibited by verapamil which also suppressed the 3H‐thymidine incorporation. The results are in keeping with the interpretation that the inhibition of T cell stimulation by TFP was not due to suppression of Ca2+ uptake, but due to inactivation of Ca2+‐calmodulin complex which might be formed subsequent to Ca2+ entry into the cell.

Mechanisms of Interleukin-2-Mediated Signaling and role of Calcium in T cell Mitogenesis

In T lymphocytes stimulated by concanavalin A (Con A), interleukin 2 (IL-2) acts on the late G1 stage of the cell cycle. Ca2+ uptake by T cells was not enhanced with the stimulation of Con A (initiation) or IL-2 (late G1 stage), but Ca2+ requirement was observed at the two stages. These results indicate that the enhancement of Ca2+ uptake is not necessary, but intracellular Ca2+ may act as an important messenger in T cell mitogenesis.

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.

Association of a cytoplasmic dynein-like protein recognizable by anti-map 1C with the mammalian mitotic spindle

A rabbit antibody to bovine brain MAP 1C was prepared. The antibody stained the mitotic spindle of PtK2 cells by immunofluorescence. On immunoblots of PtK2 cell extract the antibody reacted with polypeptides of molecular weights greater than 350 and 80 KD that resemble the subunit proteins of bovine brain MAP 1C. An additional 135 KD polypeptide in the extract was also stained. These results indicate that a cytoplasmic dynein recognizable by the anti-MAP 1C antibody is localized in the mitotic spindle.

Seasonal variation in mitogenic response and subsets of human tonsillar lymphocytes

Abstract A seasonal variation was observed in the mitogenic response of human tonsillar lymphocytes and in the percentages of each subset. We found that the responsiveness of lymphocytes to mitogens was high in summer and low in winter. The identification of lymphocyte subsets by using monoclonal antibodies indicated that the percentages of T4 positive cells and T8 positive cells increased in summer and decreased in winter. The most significant change was observed in the percentages of T8 positive cells. The significant relationship between the mitogenesis and the percentages of T8 positive cells was observed. These results indicated that the seasonal variation in lymphocyte mitogenesis might depend on the size of T8 positive cells.

Analysis of mammalian dynein using antibodies against A polypeptides of sea urchin sperm flagellar dynein.

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.

Rare chromosomal aberrations induced by vincristine: Partial endoreduplication and pseudoendoreduplication, segmentally endoreduplicated chromosomes, and segmental premature chromosome condensation

Vincristine (VCR) is capable of inducing a cell containing both conventional chromosomes (monochromosomes) and diplochromosomes. A total of 124 such metaphases were examined by 5-bromodeoxyuridine (BrdU) incorporation and fluorescence plus Giemsa (FPG) technique to analyze cell cycle kinetics. The majority of cells (119 metaphases) showed differential BrdU incorporation between the two kinds of chromosomes, indicating that partial endoreduplication occurred in these cells. In addition, existence of partially endoreduplicated cells with premature chromosome condensation (PCC) in either mono- or diplochromosomes suggests that the timing of monochromosome-replication was very variable in individual cells. On the other hand, the remaining five metaphases showed that both mono- and diplochromosomes incorporated BrdU similarly, indicating that diplochromosomes are formed by pseudoendoreduplication. Two kinds of chromosomal aberrations probably caused by delay of DNA synthesis on chromosome segments, segmental endoreduplication, and segmental PCC were also reported. Segmental endoreduplication was defined as endoreduplication that occurred on some segments of chromosomes. Out of 119 partially endoreduplicated cells, 3 contained a chromosome consisting of both mono- and diplochromosomal segments, indicating that the former segments missed one round of DNA synthesis. Segmental PCC was defined as PCC restricted to only some segments of chromosomes. Two types of segmental PCC, segmental S-PCC and G2-PCC, were observed in VCR-induced ordinary polyploidy. Although both segmental endoreduplication and segmental PCC occurred with very low frequency, these phenomena suggest that DNA synthesis was disturbed in some part of the nucleus.