Development of compartment for studies on the growth of protein crystals in space.

To clarify the growth mechanism of a protein crystal, it is essential to measure its growth rate with respect to the supersaturation. We developed a compartment (growth cell) for measuring the growth rate (<0.1 nm s(-1)) of the face of a protein crystal at a controlled supersaturation by interferometry over a period of half a year in space. The growth cell mainly consists of quartz glass, in which the growth solution and a seed crystal are enclosed by capillaries, the screw sample holder, and a helical insert. To avoid the destruction of the cell and the evaporation of the water from the solution inside the cell, we selected the materials for these components with care. The equipment was successfully used to examine the growth of a lysozyme crystal at a controlled supersaturation in space, where convection is negligible because of the microgravity environment, thereby advancing our understanding of the mechanism of protein crystal growth from solution. The technique used to develop the growth cell is useful not only for space experiments but also for kinetic studies of materials with very slow growth and dissolution rates (<10(-3) nm s(-1)).

Protein crystallization by using porous glass substrate.

The effects of a commercially available porous glass substrate (Corning Porous Glass No.7930) on the heterogeneous nucleation of proteins [hen egg-white lysozyme (HEWL), thaumatin and apoferritin] have been investigated in order to develop an improved method to facilitate the nucleation of protein crystals. It was found that the porous glass substrate could promote the nucleation at lower supersaturations. The induction time for nucleation decreased, and the crystals obtained from porous glass substrates were larger than those from normal glass substrates. Many pores and channels of 10-100 nm in diameter were observed on the porous glass surface by atomic force microscopy (AFM). It is believed that these pores and channels are crucial for facilitating the nucleation process in this work.

A molecular biological study of anti-tumor mechanisms of an anti-cancer agent Oxaliplatin against established human gastric cancer cell lines.

We report that preoperative administration of Oxaliplatin, a new anti-cancer platinum agent, is an effective treatment for gastric cancer. The purpose of this in vitro study is to determine whether Oxaliplatin induces apoptosis in established human gastric cancer cell lines. Five established gastric cancer cell lines are used: MNK45, KATO-III, OKAJIMA, MNK28 and MNK74. Chemosensitivity to l-OHP is studied using a growth inhibition test. Induction of apoptosis in gastric cancer cells is analyzed by assessing DNA ladder formation, DNA fragmentation and actin cleavage. While all five gastric cancer cell lines are sensitive to Oxaliplatin, the poorly differentiated lines are the most sensitive. DNA ladder formation and/or DNA fragmentation are detected in all gastric cancer cell lines. However, actin cleavage is not detected in any of the cell lines. Oxaliplatin has an anti-cancer effect on human gastric cancer cell lines, particularly cell lines of poorly differentiated adenocarcinoma, indicating that Oxaliplatin would be an effective treatment for poorly differentiated gastric cancer. Oxaliplatin induces apoptosis in gastric cancer cell lines, but actin cleavage is not detected in cancer cells. This finding suggests that (1) the apoptotic caspase pathway leads mainly to DNA condensation and fragmentation, and (2) caspase-independent apoptotic pathways may be activated when gastric cancer cells are treated with Oxaliplatin.

Does the timing of surgery for breast cancer in relation to the menstrual cycle or geomagnetic activity affect prognoses of premenopausal patients?

We examined the records in 36 breast cancer patients treated between 1990 and 2001, and compared them for relapse-free survival with reference to the phases of menstrual cycle defined by Hrushesky et al. and Senie et al. During the follow-up period, seven patients suffered a relapse and one died of another disease without relapsed breast cancer. The recurrence rate and relapse-free survival were not significantly different with the menstrual timing of surgery. However, patients with early breast cancer operated during the follicular phase and those with advanced breast cancer resected during the luteal phase appeared to show better prognosis than corresponding controls operated during the other phases. On the other hand, the correlation between geomagnetic activity and prognosis of breast cancer was also investigated. High geomagnetic activity during operation significantly affected the prognosis of the disease in an adverse fashion. This adverse influence was more marked in the patients operated during the luteal period. Since the menstrual cycle has no clear relation to the prognosis of breast cancer, the geomagnetic activity might affect them via other pathways than the menstrual cycle.

Inhibition of growth of human breast cancer cells in culture by neutron capture using liposomes containing 10B.

Cell destruction in boron neutron capture therapy is effected by nuclear reaction between 10B and thermal neutrons with the release of alpha-particles (4He) and lithium-7 ions (7Li). 4He kills cells within 10 microm of the site of 4He generation, therefore it is theoretically possible to destroy tumour cells without affecting adjacent healthy tissue, given selective delivery of compounds containing 10B. Liposomes wore prepared by vortex dispersion of solutions containing 10B compounds with dried lipid films and the effects of those compounds on human breast cancer cells in culture were examined after thermal neutral irradiation. [3H]-TdR incorporation by MRKnu/nu-1 cells treated with 10B-containing liposomes showed 40% suppression compared with liposomes without 10B, at 2 x 1012 n/cm2 thermal neutron fluence. Inhibition of tumour cell growth with liposomes prepared with 100 mm 10B-compound was as significant as with those made with 500 ppm 10B solution. The concentration of 10B in liposomes was 76.5 +/- 3.4 microg/mL. Boronated liposomes can thus deliver sufficient 10B atoms to this line of breast cancer cells in culture to effect cytotoxicity and suppression of growth after thermal neutron irradiation.

Docetaxel alone or orally combined with 5-fluorouracil and its derivatives: effects on mouse mammary tumor cell line MM2 in vitro and in vivo.

Although docetaxel (Taxotere; TXT), a taxoid anticancer drug, is clinically and experimentally very effective against breast cancer, its antitumor effect is of very short duration. We addressed whether 5-fluorouracil (5-FU) and its derivatives can act synergistically with TXT against mammary tumors, with placing particular stress on their use by oral route. Mouse mammary tumor cell line, MM2, was propagated in culture and as ascites in mice. Carmofur (HCFU) and doxifluridine (5'-DFUR) were used as 5-FU derivatives. In vitro, the cytotoxic effects of antitumor drugs on MM2 cells were examined by MTS assay. In vivo, mice inoculated i.p. with MM2 cells were treated with i.p. injection of TXT and/or oral administration of 5-FU or its derivatives, and observed for curing tumor. In vitro, the synergistic effects were observed in the combination of TXT and 5-FU or HCFU, but not in that of TXT and 5'-DFUR. In vivo, all of these combinations cured tumors far more effectively than TXT alone. The discrepant result of the combination of TXT and 5'-DFUR between in vitro and in vivo was ascribed to up-regulation of pyrimidine phosphorylase in tumor cells in vivo by TXT. Thus, 5-FU, its masked compounds like HCFU and its prodrugs like 5'-DFUR can act synergistically with TXT in the therapy of cancer even when administered by the oral route.

Systematic analysis of supersaturation and lysozyme crystal quality.

A systematic study of the correlation between supersaturation and protein crystal quality was carried out employing atomic force microscopy (AFM) and X-ray crystallography with synchrotron radiation (SR). The surface morphology and growth rates of hen egg-white (HEW) lysozyme crystals soaked in various supersaturated solutions were first investigated by AFM. The results showed that the formation of two-dimensional islands increased as a function of supersaturation. The growth rate (molecule intake speed) also increased as a function of supersaturation. In order to examine the correlation between the surface morphology, growth rate and the crystal quality, X-ray diffraction experiments were performed. It was confirmed that crystals grown at lower supersaturations diffracted better with higher signal-to-noise ratios, including better agreement between symmetry-related reflections. The results strongly suggested that the molecular misorientation at high supersaturation affected the crystal quality.

Correlation between timing of surgery in relation to the menstrual cycle and prognosis of premenopausal breast cancer patients.

The timing of surgery in relation to menstrual phase might affect the progress of disease in premenopausal women with operable breast cancer. In the present study, the records were examined of 28 such cases treated between 1990 and 1999, and compared for recurrence-free survival with reference to the phases of the menstrual cycle defined by Hrushesky and Senie. During the follow-up period, breast cancer relapse occurred in five patients, and one patient died of another disease unconnected with recurrent breast cancer. The recurrence rate was not significantly different between two phases classified by either Hrushesky or Senie. However, patients with early-stage breast cancer operated during the perimenstrual phase and those with advanced breast cancer which was resected during the peri-ovulatory phase appeared to have a better prognosis than patients operated on during the other phases. Since the prognosis for breast cancer patients is dependent not only on the menstrual cycle but also on many other factors, it is concluded that the menstrual cycle cannot constitute an absolute prognostic factor.

Morphogenesis in cucumber seedlings is negatively controlled by gravity.

Seedlings of most cucurbitaceous plants develop a peg (protuberance caused by cell outgrowth) on the transition zone between the hypocotyl and root. The peg is necessary for removing the seed coat after germination. In our spaceflight experiments on the STS-95 space shuttle, Discovery, we found that cucumber (Cucumis sativus L.) seedlings grown under microgravity conditions developed two pegs symmetrically at the transition zone. Thus, cucumber seedlings potentially develop two pegs and do not require gravity for peg formation itself, but on the ground the development of one peg is suppressed in response to gravity. This may be considered as negative control of morphogenesis by gravity.

STS-95 space experiment for plant growth and development, and auxin polar transport.

The principal objective of the space experiment, BRIC-AUX on STS-95, was the integrated analysis of the growth and development of etiolated pea and maize seedlings in space, and the effect of microgravity conditions in space on auxin polar transport in the segments. Microgravity conditions in space strongly affected the growth and development of etiolated pea and maize seedlings. Etiolated pea and maize seedlings were leaned and curved during space flight, respectively. Finally the growth inhibition of these seedlings was also observed. Roots of some pea seedlings grew toward the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles which were germinated and grown under microgravity conditions in space were significantly low. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and extremely promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.

Inhibition of root elongation in microgravity by an applied electric field.

Roots grown in an applied electric field demonstrate a bidirectional curvature. To further understand the nature of this response and its implications for the regulation of differential growth, we applied an electric field to roots growing in microgravity. We found that growth rates of roots in microgravity were higher than growth rates of ground controls. Immediately upon application of the electric field, root elongation was inhibited. We interpret this result as an indication that, in the absence of a gravity stimulus, the sensitivity of the root to an applied electric stimulus is increased. Further space experiments are required to determine the extent to which this sensitivity is shifted. The implications of this result are discussed in relation to gravitropic signaling and the regulation of differential cell elongation in the root.

Gravimorphogenesis of Cucurbitaceae plants: development of peg cells and graviperception mechanism in cucumber seedlings.

We examined the effect of microgravity on the peg formation of cucumber seedlings for clarifying the mechanism of gravimorphogenesis in cucurbitaceous plants. The spaceflight experiments verified that gravity controls the formation of peg, hypocotyl hook and growth orientation of cucumber seedlings. Space-grown cucumber developed a peg on each side of the transition zone of the hypocotyl and root, indicating that on the ground peg formation is regulated negatively by gravity (Takahashi et al. 2000). It was found that the auxin-regulated gene, CS-IAA1, was strongly expressed in the transition zone where peg develops (Fujii et al. 2000). In the seedlings grown horizontally on the ground, CS-IAA1 transcripts were much abundant on the lower side of the transition zone, but no such differential expression of CS-IAA1 was observed in the space-grown cucumber (Kamada et al. 2000). These results imply that gravity plays a role in peg formation through auxin redistribution. By the negative control, peg formation on the upper side of the transition zone in the horizontally growing seedlings might be suppressed due to a reduction in auxin concentration. The threshold theory of auxin concentration accounted for the new concept, negative control of morphogenesis by gravity (Kamada et al. 2000). Anatomical studies have shown that there exists the target cells destined to be a peg and distinguishable at the early stage of the growth. Ultra-structural analysis suggested that endoplasmic reticulum develops well in the cells of the future peg. Furthermore, it was found that reorganization of cortical microtubules is required for the change in cell growth polarity in the process of peg formation. The spaceflight experiment with cucumber seedlings also suggested that in microgravity positive hydrotropic response of roots occurred without interference by gravitropic response (Takahashi et al. 1999b). Thus, this spaceflight experiment together with the ground-based studies has shown that cucumber seedling is an ideal for the study of gravimorphogenesis, hydrotropism and their interaction. Although peg formation is seen specifically in cucurbitaceous seedlings, it involves graviperception, auxin transport and redistribution and cytoskeletal modification for controlling cell growth polarity. This system could be a useful model for studying important current issues in plant biology.

Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism.

During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.

Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment.

The principal objectives of the space experiment, BRIC-AUX on STS 95, were the integrated analysis of the growth and development of etiolated pea and maize seedlings in space and a study of the effects of microgravity conditions in space on auxin polar transport in these segments. Microgravity significantly affected the growth and development of etiolated pea and maize seedlings. Epicotyls of etiolated pea seedlings were the most oriented toward about 40 to 60 degrees from the vertical. Mesocotyls of etiolated maize seedlings were curved at random during space flight but coleoptiles were almost straight. Finally the growth inhibition of these seedlings in space was also observed. Roots of some pea seedlings grew toward to the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles, which were germinated and grown under microgravity conditions in space, were significantly low as compared with those grown on the ground of the earth. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.

Morphogenesis of rice and Arabidopsis seedlings in space.

Seedlings of rice (Oryza saliva L.) and Arabidopsis (A. thaliana L.) were cultivated for 68.5 hr in the RICE experiment on board during Space Shuttle STS 95 mission, and changes in their growth and morphology were analyzed. Microgravity in space stimulated elongation growth of both rice coleoptiles and Arabidopsis hypocotyls by making their cell walls extensible. In space, rice coleoptiles showed an inclination toward the caryopsis in the basal region and also a spontaneous curvature in the same direction in the elongating region. These inclinations and curvatures were more prominent in the Koshihikari cultivar compared to a dwarf cultivar, Tan-ginbozu. Rice roots elongated in various directions including into the air on orbit, but two thirds of the roots formed a constant angle with the axis of the caryopsis. In space, Arabidopsis hypocotyls also elongated in a variety of directions and about 10% of the hypocotyls grew into the agar medium. No clear curvatures were observed in the elongating region of Arabidopsis hypocotyls. Such a morphology of both types of seedlings was fundamentally similar to that observed on a 3 D clinostat. Thus, it was confirmed by the RICE experiment that rice and Arabidopsis seedlings perform an automorphogenesis under not only simulated but also true microgravity conditions.

Inhibition of root elongation in microgravity by an applied electric field.

Roots grown in an applied electric field demonstrate a bidirectional curvature. To further understand the nature of this response and its implications for the regulation of differential growth, we applied an electric field to roots growing in microgravity. We found that growth rates of roots in microgravity were higher than growth rates of ground controls. Immediately upon application of the electric field, root elongation was inhibited. We interpret this result as an indication that, in the absence of a gravity stimulus, the sensitivity of the root to an applied electric stimulus is increased. Further space experiments are required to determine the extent to which this sensitivity is shifted. The implications of this result are discussed in relation to gravitropic signaling and the regulation of differential cell elongation in the root.

Spatial expression of Drosophila glutathione S-transferase-D1 in the alimentary canal is regulated by the overlying visceral mesoderm.

Regional gene expression within Drosophila gut epithelium is regulated by the homeotic genes expressed in the overlying visceral mesoderm. Here it is reported that Glutathione S-transferase-D1 (Gst-D1) had three distinctive expression domains in the gut epithelia: the inner epithelium of the proventriculus, the anterior border of the hindgut epithelium, and the midgut epithelium. Gst-D1 expression in the midgut epithelium became restricted to the region that later formed the third midgut constriction. This spatial restriction within the midgut epithelium required abdominal-A activity in the overlying visceral mesoderm, suggesting that Gst-D1 will be a useful marker to analyze the mechanism of gene regulation across the mesoderm and endoderm.

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells.

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions. Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-El cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.