Development and disintegration of phragmoplasts in living cultured cells of a GFP::TUA6 transgenic Arabidopsis thaliana plant.

Cultured suspension cells of Arabidopsis thaliana that stably express a green-fluorescent protein-alpha-tubulin 6 fusion protein were used to follow the development and disintegration of phragmoplasts. The development and disintegration of phragmoplasts in the living cultured cells could be successively observed by detecting the green-fluorescent protein fluorescence of the microtubules. In the early telophase spindle, where two kinetochore groups and two daughter chromosome groups had completely separated from one another, fluorescence appeared in the interzone between the two chromosome groups. The fluorescent region was gradually condensed at the previous equator and increased in fluorescence intensity, and finally it formed the initial phragmoplast. The initial phragmoplast moved from the cell center towards the cell periphery, and it lost fluorescence at its center and became double rings in shape. The expansion orientation of the phragmoplast was not always the same as that of the future new cell wall before it came in contact with the cell wall. The phragmoplast did not usually come in contact with the cell wall simultaneously with its entire length. A portion of the phragmoplast which was earlier in contact with the cell wall disappeared earlier than other portions of the phragmoplast. The duration of contact between any portions of the phragmoplast and the plasma membrane of the cell wall was 15-30 min. The fluorescence intensity of the cytoplasm did not seem to be elevated by the disintegration of the strongly fluorescent phragmoplast.

Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin.

The mode of cytokinesis, especially in determining the site of cell division, is not well understood in higher-plant cells. The division site appears to be predicted by the preprophase band of microtubules that develop with the phragmosome, an intracellular structure of the cytoplasm suspending the nucleus and the mitotic apparatus in the center. As the preprophase band disappears during mitosis, it is thought to leave some form of "memory" on the plasma membrane to guide the growth of the new cell plate at cytokinesis. However, the intrinsic nature of this "memory" remains to be clarified. In addition to microtubules, microfilaments also dynamically change forms during cell cycle transition from the late G2 to the early G1 phase. We have studied the relationships between microtubules and microfilaments in tobacco BY-2 cells and transgenic BY-2 cells expressing a fusion protein of green-fluorescent protein and tubulin. At the late G2 phase, microfilaments colocalize with the preprophase band of microtubules. However, an actin-depleted zone which appears at late prometaphase is observed around the chromosomes, especially at metaphase, but also throughout anaphase. To study the functions of the actin-depleted zone, we disrupted the microfilament structures with bistheonellide A, a novel macrolide that depolymerizes microfilaments very rapidly even at low concentrations. The division planes became disorganized when the drug was added to synchronized BY-2 cells before the appearance of the actin-depleted zone. In contrast, the division planes appeared smooth, as in control cells, when the drug was added after the appearance of the actin-depleted zone. These results suggest that the actin-depleted zone may participate in the demarcation of the division site at the final stage of cell division in higher plants.

Fate of nascent microtubules organized at the M/G1 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells.

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.

Restoration of hearing with an auditory brainstem implant in a patient with neurofibromatosis type 2--case report.

A 25-year-old male with neurofibromatosis type 2 had hearing restored with an auditory brainstem implant (ABI) after removal of an acoustic schwannoma. The ABI allows the patient to discern many different environment sounds and is a significant adjunct to lip-reading, enabling conversation with people who have clear pronunciation without the necessity for writing.

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes.

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

Time-sequence observations of microtubule dynamics throughout mitosis in living cell suspensions of stable transgenic Arabidopsis--direct evidence for the origin of cortical microtubules at M/G1 interface.

Transgenic Arabidopsis thaliana, stably expressing a GFP-TUA6 fusion protein, were subcultured in B5 medium supplemented with 2,4-D and BA. In the cell suspensions, the microtubular changes in the mitotic cells could be monitored by time-sequence observations using a time-lapse system of fluorescence microscopy. We have succeeded in following the microtubule (MT) dynamics in living cells throughout mitosis, from the late G2 phase to early G1 phase, and found that, at the M/G1 interface, the cortical MTs were firstly reorganized in the perinuclear regions and then in the cortex, as we had previously suggested (Hasezawa and Nagata 1991, Nagata et al. 1994). The significance of this observation on the origin of cortical MTs is discussed.

Putative involvement of a 49 kDa protein in microtubule assembly in vitro.

In higher plant cells, thus far only a few molecules have been inferred to be involved in microtubule organizing centers (MTOCs). Examination of a 49 kDa tobacco protein, homologous to a 51 kDa protein involved in sea urchin MTOCs, showed that it also accumulated at the putative MTOC sites in tobacco BY-2 cells. In this report, we show that the 49 kDa protein is likely to play a significant role in microtubule organization in vitro. We have established a system prepared from BY-2 cells, capable of organizing microtubules in vitro. The fraction, which was partially purified from homogenized miniprotoplasts (evacuolated protoplasts) by salt extraction and subsequent ion exchange chromatography, contained many particles of diameters about 1 micron after desalting by dialysis. When this fraction was incubated with purified porcine brain tubulin, microtubules were elongated radially from the particles and organized into structures similar to the asters observed in animal cells, and therefore also termed "asters" here. Since we could hardly detect BY-2 tubulin molecules in this fraction, the microtubules in "asters" seemed to be solely composed of the added porcine tubulin. Tubulin molecules were newly polymerized at the ends of the microtubules distal to the particles, and the elongation rate of microtubules was more similar to the reported rate of the plus-ends than that of the minus-ends in vitro. By fluorescence microscopy, the 49 kDa protein was shown to be located at the particles. Thus, its location at the centers of the "asters" suggests that the protein plays a role in microtubule organization in vitro.

Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line.

Synchronous cell systems are highly desirable for investigating various aspects of plant cell biology. However, to date, the tobacco BY-2 cell line is the only plant cell line which can be synchronized to high levels. A cell synchrony starting from S phase is obtained after release of BY-2 cells from aphidicolin treatment, while that from M phase is available after release from a sequential treatment of aphidicolin followed by propyzamide. A high level of synchrony is only attained by using rapidly growing tobacco BY-2 cells that propagate ca. 100-fold in a week. Reduced levels of synchrony result if the growth rate becomes lower. Technical notes for maintaining the high growth rate of the tobacco BY-2 cell are described. Using this highly synchronous system it has been possible to demonstrate the cell cycle-dependent oscillation of many genes, such as cyclins, and characterize their role during the cell cycle. Furthermore, this system has facilitated the structural and biochemical analysis of cell cycle specific events such as the development of the phragmoplast and the formation of cortical microtubules. Other potential uses of this highly synchronized cells are also described.

Carcinosarcoma of the ureter with unusual histologic features.

We report a rare case of carcinosarcoma of the ureter presented as a retroperitoneal tumor. The tumor originated in the ureter as a polypoid protrusion and spread around peri-ureteral, retroperitoneal tissues. The polypoid ureteral tumor was composed of an intra-epithelial carcinoma and a submucosal mesenchymal tumor; the histology of the retroperitoneal tumor in the original site was that of a carcinosarcoma. The bulk of the tumor in the retroperitoneum was composed of blastematous, epithelial and sarcomatous neoplastic cells with foci resembling an incomplete glomeruloid formation, thus mimicking a Wilms' tumor. The lesion appeared to originate from multipotential cells in the mucosal layer of the ureter. Whether or not this tumor has a true nephrogenic property is currently unknown. When a retroperitoneal tumor of the adult resembling a Wilms' tumor is found, one should suspect a possible carcinosarcomatous origin in the ureter.

[Hybrid bioartificial liver using canine hepatocytes in primary culture].

Viable Hepatocytes were isolated from adult canine liver by in situ collagenase perfusion, and cultured on collagen coated borosilicate glass plates (100 X 200mm) at confluent cell density. The medium of hepatocytes in the primary culture was L-15 supplemented with aprotinin 5000U/L, proline 30mg/L, insulin 10(-8)M, dexamethasone 10(-8)M, glucagon 10(-8)M, and h-EGF 10ng/ml. Long-stroke type bioartificial liver module consisted of 200 glass plates with hepatocytes. It contained 6 billion primary cultured cells in total, that is almost equivalent to 30% of the normal canine liver. All hepatocytes in the module were quite viable during 2 weeks in the perfusion culture, and maintained various liver functions at a high level. Gluconeogenesis was 368.0 +/- 15.4mg/module/hr, albumin synthesis was 19.1 +/- 2.5mg/module/day, ureogenesis was 3.7 +/- 0.1mg/module/hr, and ammonia metabolism was 8.4mg/module/hr. Moreover, those functions were maintained at least 2 weeks in the canine plasma as well as in the culture medium with hormones. This hybrid bioartificial liver may exert various liver functions like a liver in situ.

A hybrid bioartificial liver composed of multiplated hepatocyte monolayers.

Monolayer cultures of hepatocytes were shown to have good function when compared with suspended cells. The authors manufactured a new hybrid artificial liver containing hepatocyte monolayers and evaluated its function. Hepatocytes isolated from an adult dog liver were cultured on collagen coated borosilicated glass (10 X 20 X 0.04 cm). A long-stroke artificial liver module was constructed by stacking 200 glass plates bearing hepatocytes, which were viable and functioned well during 4 weeks in perfusion culture; glyconeogenesis = 110 ng/micrograms DNA/min, urea synthesis = 3.6 ng/micrograms DNA/min and albumin synthesis = 29 micrograms/10(6) cells/day at the 5th day of perfusion. The levels were maintained for 2 weeks. The new device was applied to anhepatic dogs (Group 3) and compared with untreated (Group 1) and plasma exchange dogs (Group 2). The survival times were 21.3 +/- 5.6 hours in Group 1 (N = 6), 27.8 +/- 4.0 hours in Group 2 (N = 3), and 55.0 +/- 10.3 hours in Group 3 (N = 4). The longest survival was 65 hours. Serum ammonia increased to over 2,000 micrograms/dl after 12 hours in Groups 1 and 2, but remained under 400 micrograms/dl in Group 3. This new type of hybrid system may be a pilot design for the complete artificial liver.

Satisfaction among rural and urban Japanese elderly in three-generation families.

For the purpose of testing an assumption on the dual nature of aging in Japanese society, this study compared and contrasted three-generation adult families in Yamato-machi, a rural town in Niigata Prefecture in northern Japan, and in Setagaya-ku, an urban ward in Tokyo. The average ages for each generation in the study were G1 = 85, G2 = 60, and G3 = 35. The findings reveal regional variation in basic demographic characteristics such as population density, family size, proportions of the elderly 65 and over and 90 and over, as well as the prevalence of the three-generation family households. This supports the existence of dual patterns of aging in Japan today. The data also show significant differences between these regional sectors in the level of educational and economic conditions of the elderly. However, measures of the extent of satisfaction do not reveal any significant difference between rural and urban elderly. It is suspected that this is because only three-generation healthy families were interviewed in this study.

Lack of effect of levallorphan on analgesia induced by intraventricular application of porcine calcitonin in mice.

Intraventricular administration to mice porcine calcitonin (10 U/kg) as well as of morphine (3 microgram/kg) elevated the threshold pressure of stimuli applied to the base of the tail as assessed by squeaking, struggling or biting, all of which were regarded as manifestations of pain sensation in the animals. Pretreatment with an opiate antagonist, levallorphan (30 mg/kg i.p.) showed no influence upon the analgesic effect of calcitonin, though it completely antagonized the effect of morphine. The results suggested that a peptide hormone, calcitonin, exerted its analgesic action in a manner distinct from the narcotic analgesic.