Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7

CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.     

Protein kinase Cβ isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells

The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3–5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCα/β. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCα or PKCβ. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCβ-treated cells, but those in antisense PKCα-treated cells were not attenuated. These suggested that PKCβ plays a regulatory role in the expression of MDR3.     

Analysis of gender difference of cardiac risk biomarkers using hGH-transgenic mice.

We investigated gender difference in the effects of chronic exposure to human growth hormone (hGH) on cardiac risk biomarkers using transgenic mice with non-pulsatile circulating hGH. Blood plasma was obtained from transgenic and control mice at 8, 12, and 16 weeks of age, and was used for the measurement of hGH and the following cardiac risk biomarkers: total cholesterol (CHO), triglyceride (TG), HDL cholesterol (HDL), LDL cholesterol (LDL), non esterified free fatty acids (NEFA), and lipid peroxides (LPO). The hearts and the livers of transgenic mice were weighed and histopathologically examined, and the results were compared with those of control mice. Transgenic males exhibited higher levels of LDL at 8 and 12 weeks of age and higher levels of LPO at every week of age examined, as compared to those of the control males, while transgenic females exhibited somewhat lower levels of LDL and LPO from 8 to 16 weeks of age, as compared to the control females. The relative heart weight in males increased with aging and was significantly higher in the 16-week-old transgenic males compared to those of the control mice. The present results demonstrate that transgenic males had cardiac risk potential caused by chronic-exposure to hGH as compared to females. The results also show that the present transgenic mouse line is a useful model for the study of gender difference in cardiac disorders caused by hGH.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Effects of Silicon-Limitation on Growth and Morphology of Triparma laevis NIES-2565 (Parmales,Heterokontophyta)

The order Parmales (Heterokontophyta) is a group of small-sized unicellular marine phytoplankton, which is distributed widely from tropical to polar waters. The cells of Parmales are surrounded by a distinctive cell wall, which consists of several siliceous plates fitting edge to edge. Phylogenetic and morphological analyses suggest that Parmales is one of the key organisms for elucidating the evolutionary origin of Bacillariophyceae (diatoms), the most successful heterokontophyta. The effects of silicon-limitation on growth and morphogenesis of plates were studied using a strain of Triparma laevis NIES-2565, which was cultured for the first time in artificial sea water. The cells of T. laevis were surrounded by eight plates when grown with sufficient silicon. However, plate formation became incomplete when cells were cultured in a medium containing low silicate (ca. <10 µM). Cells finally lost almost all plates in a medium containing silicate concentrations lower than ca. 1 µM. However, silicon-limitation did not affect growth rate; cells continued to divide without changing their growth rate, even after all plates were lost. Loss of plates was reversible; when cells without plates were transferred to a medium containing sufficient silicate, regeneration of shield and ventral plates was followed by the formation of girdle and triradiate plates. The results indicate that the response to silicon-limitation of T. laevis is different from that of diatoms, where cell division becomes inhibited under such conditions.     

Structural basis for oligosaccharide recognition of misfolded glycoproteins by OS-9 in ER-associated degradation

Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6-phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost α1,2-linked mannose on C-arm of high-mannose-type glycan and binding of processed α1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9(MRH)) complexed with α3,α6-mannopentaose. The OS-9(MRH) has a flattened β-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural insights into the mechanism whereby OS-9 specifically recognizes Manα1,6Manα1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif.     

Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250°C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130°C for 60 min or at 140°C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130°C for 60 min or at 140°C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.Endotoxins are lipopolysaccharides (LPS) that are derived from the cell membranes of gram-negative bacteria and are continuously released into the environment. The release of LPS occurs not only upon cell death but also during growth and division. In the pharmaceutical industry, it is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities, including pyrogenicity, lethality, Schwartzman reactivity, adjuvant activity, and macrophage activation (2, 9, 12, 13, 25, 32). Endotoxins are very stable molecules that are capable of resisting extreme temperatures and pH values (3, 16, 17, 29, 30, 34, 38). An endotoxin monomer has a molar mass of 10 to 20 kDa and forms supramolecular aggregates in aqueous solutions (22, 39) due to its amphipathic structure, which makes depyrogenation more difficult than sterilization. Endotoxins are not efficiently inactivated with the regular heat sterilization procedures recommended by the Japanese Pharmacopoeia. These procedures are steam heat treatment at 121°C for 20 min or dry-heat treatment for at least 1 h at 180°C. It is well accepted that only dry-heat treatment is efficient in destroying endotoxins (3, 16, 29, 30) and that endotoxins can be inactivated when exposed to a temperature of 250°C for more than 30 min or 180°C for more than 3 h (14, 36). In the production of parenterals, it is necessary to both depyrogenate the final products and carry out sterilization to avoid bacterial contamination.Several studies have examined dry-heat treatment, which is a very efficient means to degrade endotoxins (6, 20, 21, 26, 41, 42). However, its application is restricted to steel and glass implements that can tolerate high temperatures of >250°C. For sterilization, dry heat treatment tends to be used only with thermostable materials that cannot be sterilized by steam heat treatment (autoclaving). Alternative depyrogenation processes include the application of activated carbon (35), oxidation (15), and acidic or alkaline reagents (27), but steam heat treatment would be an attractive option if it were sufficiently effective. However, the data on the inactivation of endotoxins by steam heat treatment are insufficient and contradictory. It has been reported that endotoxins were not efficiently inactivated by steam heat treatment at 121°C (19, 45). However, Ogawa et al. (31) recently reported that steam heat treatment was efficient in inactivating low concentrations of endotoxin, and that Escherichia coli LPS are unstable in aqueous solutions even at relatively low temperatures such as 70°C (see also reference 40). As mentioned above, these reports have shown that although studies have been carried out on the use of steam heat for depyrogenation, there is little agreement on its efficiency.The U.S. Pharmacopoeia (USP) recommends depyrogenation by dry-heat treatment at temperatures above 220°C for as long as is necessary to achieve a ≥3-log reduction in the activity of endotoxin, if the value is ≥1,000 endotoxin units (EU)/ml (11, 44). Due to the serious risks associated with endotoxins, the U.S. Food and Drug Administration (FDA) has set guidelines for medical devices and parenterals. The protocol to test for endotoxin contamination of medical devices recommends immersion of the device in endotoxin-free water for at least 1 h at room temperature, followed by testing of this extract/eluate for endotoxin. Current FDA limits are such that eluates from medical devices may not exceed 0.5 EU/ml, or 0.06 EU/ml if the device comes into contact with cerebrospinal fluid (43). The term EU describes the biological activity of endotoxins. For example, 100 pg of the standard endotoxin EC-5, 200 pg of EC-2, and 120 pg of endotoxin from E. coli O111:B4 all have an activity of 1 EU (17, 23).Steam heat treatment is comparatively easy to apply and control. If steam heat treatment could reliably inactivate endotoxins, it could be applied with sterilization, reducing labor, time, and expenditure. However, to our knowledge, few studies have addressed steam heat inactivation to determine the chemical and physical reactions that occur during the hydrothermal process, nor have any studies examined the relationship between the steam saturation ratio and the inactivation of endotoxins. Moreover, to date no study has been conducted on steam heat activation of endotoxins with reference to the chemical and physical parameters of the hydrothermal process.We have developed a groundbreaking method to thermoinactivate endotoxins by means of a soft hydrothermal process, in which the steam saturation ratio can be controlled. The steam saturation ratio is calculated as follows: steam saturation ratio (%) = [steam density (kg/m³)/saturated steam density (kg/m³)] × 100.The soft hydrothermal process lies in the part of the liquid phase of water with a high steam saturation ratio that is characterized by a higher ionic product (k_w) than that of ordinary water. The ionic product is a key parameter in promoting ionic reactions and can be related to hydrolysis. The ionic product of water is 1.0 × 10⁻¹⁴ (mol/liter)² at room temperature and increases with increasing temperature and pressure. A high ionic product favors the solubility of highly polar and ionic compounds, creating the possibility of accelerating the hydrolysis reaction process of organic compounds. Thus, water can play the role of both an acidic and an alkaline catalyst in the hydrothermal process (Fig. ​(Fig.1)1) (1, 37, 46). However, the soft hydrothermal process lies in the high-density water molecular area of the steam-gas biphasic field (Fig. ​(Fig.1)1) and is characterized by a lower dielectric constant (ɛ) than that of ordinary water. This process opens the possibility of promoting the affinity of water for nonpolar or low-polarity compounds, such as lipophilic organic compounds (46). We previously reported that most of the predominant aromatic hydrocarbons were removed from softwood bedding that had been treated by soft hydrothermal processing (24, 28).Open in a separate windowFIG. 1.Reaction field in the pressure-temperature relationship of water. The curve represents the saturated vapor pressure curve. The fields show where the pressure-temperature relationships are conducive to a variety of hydrothermal processing conditions, in which water has a large impact as a reaction medium. Because high-density water has a large dielectric constant and ionic product, it is an effective reaction medium for advancing ionic reactions, whereas water (in the form of steam) on the lower-pressure side of the saturated vapor pressure curve shows a good ability to form materials by covalent bonding. Small changes in the density of water can result in changes in the chemical affinity, which has the potential to advance a range of ionic and radical reactions.The purpose of the present study was to evaluate the thermoinactivation of endotoxins by the soft hydrothermal process, by controlling the steam saturation ratio, temperature, and time of treatment. There have been reports that endotoxins were thermoinactivated by steam heat treatment at 121°C in the presence of a nonionic surfactant and at over 135°C in its absence (4, 5, 10), but the minimum temperature for the inactivation of endotoxin remained unknown. This report provides the answer to this question.     

Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents

C-3 esterifications of betulinic acid (BA, 1) and its A-ring homolog, ceanothic acid (CA, 2), were carried out to provide sixteen terpenoids, 4–19, including nine new compounds (4–12). All synthesized compounds were evaluated in an in vitro antitumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among them, compounds 4–6, 11–14, 16, and 17 displayed remarkable inhibitory effects of EBV-EA activation. BA analog 6, which contains a prenyl-like group, showed the most potent inhibitory effect (100%, 76%, 37%, and 11% inhibition of EBA activation at 1000, 500, 100, and 10 mol ratio/TPA, respectively, with IC₅₀ value of 285 mol ratio/32 pmol TPA). Compound 6 merits further development as a cancer preventive agent.     

Genetic diversity,structure, and demography of Pandanus boninensis (Pandanaceae) with sea drifted seeds,endemic to the Ogasawara Islands of Japan: Comparison between young and old islands

Pandanus boninensis, endemic to the Ogasawara Islands, Japan, is distributed on both the older Bonin and younger Volcano Islands. In this study, we conducted population genetic analyses of P. boninensis on these islands to examine the population diversity and structure across old and young islands, to assess potential differences in population demography with island age, and to collect any evidence of migration between old and young islands. We found that the genetic diversity of expressed sequence tag (EST)–based microsatellite (SSR) markers, the nucleotide diversity of nuclear DNA sequences, and the haplotype diversity of chloroplast DNA on young islands were lower than those on old islands. Clustering analyses of EST‐SSR indicated that populations on old islands were strongly diverged from those on young islands. Approximate Bayesian computation analysis of EST‐SSR suggested that population expansion occurred on old islands while population reduction occurred on young islands. We also found evidence of migration among old islands (mostly from south to north), while it appears that there have been very few migration events between old and young islands. These differences could be due to the fact that young islands tend to be geographically isolated and support smaller populations that began a shorter time ago from limited founders. The P. boninensis populations on the Volcano Islands are interesting from an evolutionary perspective as they constitute a classic example of the early stages of progressive colonization on oceanic islands with small effective population sizes and low genetic diversity.