Hideki Takanari

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.

Replication sites as revealed by double label immunofluorescence against proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) in synchronized CHO cells and vincristine-induced multinucleate cells

Summary— Double label immunofluorescence against PCNA and BrdU clearly revealed several characteristics of DNA replication sites in synchronized CHO cells. We observed that the distribution of replication sites changed from early to late S phase, particularly in the nucleolar and perinuclear regions, and the amount of PCNA in each replication site markedly decreased or disappeared with the progression of S phase. Although co‐localization of PCNA and BrdU was usually seen, the intensity of fluorescence occasionally differed between the labeled PCNA and BrdU, particularly in late S phase. Based on the assumption that such a difference may reflect a different configuration of the chromatin, we propose that the brightly granular fluorescent staining of PCNA or BrdU in early S phase indicates a condensed segment of euchromatin. In the late S phase nucleus, we clearly observed a chromatin‐like structure in the late replicating segments of heterochromatin in anti‐PCNA stained material. In vincristine‐induced multinucleate cells, a discrepancy between PCNA‐distribution and BrdU‐incorporation in sister nuclei was sometimes seen. Such observations indirectly support the following two mechanisms for premature chromosome condensation proposed by others: asynchronous initiation of DNA synthesis; and a difference in the rate of DNA synthesis. In addition, the finding that BrdU was not incorporated into sites that had PCNA deposits suggests a third mechanism: the local disturbance of replication sites. Furthermore, unusual distribution patterns for replication sites suggest that the nucleation process in multinucleate cells differs from that in normal cells.

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.

Effects of acidic pH on the formation of vinblastine-induced paracrystals in Chinese hamster ovary cells

Summary— The pH‐related change in morphology of vinblastine (VLB)‐induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB‐induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHes showed that low pHis (6.7 and 6.3) suppressed paracrystal‐formation at lower concentrations of VLB (5×10−6 M and 10−5 M). At higher concentrations of VLB (5×10−5 M and 10−4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder‐like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.

A case of pulmonary sclerosing hemangioma

A case of pulmonary sclerosing hemangioma was studied using light microscopic, immunohistochemical and electron microscopic methods. The cytoplasm of the sclerosing hemangioma cells was positive to the anti-lung surfactant apoprotein monoclonal antibody (PE-10). These cells included pale cells of the solid areas, cells covering papillary projections, and cells lining cleft-like spaces. Electron microscopic study showed that the predominant cells included poorly-differentiated pneumocytes and normal Type 2 pneumocytes. We concluded that the sclerosing hemangioma was an epithelial tumor with differentiation towards Type 2 pneumocytes.