AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.     

Structural and functional evolution of three cardiac natriuretic peptides

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.     

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.     

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.     

Membrane fluidity change in erythrocytes induced by complement system.

The structural change in erythrocyte membranes induced by antibody and complement was studied using phospholipid spin-labels. Sheep erythrocytes were labeled with phosphatidylcholine spin-label and various intermediate cells (erythrocyte-antibody complex (EA), EA bound with complement components from C1 to C7 (EAC1-7), EAC1-8, and EAC1-9) were prepared. Electron spin resonance spectra of EA, EAC1-7, and EAC1-8 were very similar to that of the erythrocytes, while that of EAC1-9 was markedly different. The overall splitting value for the lysed EAC1-9 (53 G) was much smaller than that for the erythrocytes (57 G), indicating a marked fluidization around the phosphatidylcholine label. The unlysed EAC1-9 membranes contained a limited fraction of the fluidized area. When EA was reacted with complement in the presence of 36% bovine serum albumin, the membranes were fluidized similarly to the lysed EAC1-9, although the hemolysis was largely blocked. The membranes of unlysed EAC1-9 prepared in isotonic (ethylenedinitrilo)tetraacetic acid were also fluidized, but to somewhat smaller extent. The role of C9 in the modification of erythrocyte membranes was also demonstrated using Mg2+ ghosts, which were prepared by hypotonic hemolysis in the presence of Mg2+. The membranes of Mg2+ ghost of EAC1-7 were markedly fluidized when bound with C8 and C9, but not affected by binding of C8 only. The component C8 was found to give a latent effect on the membranes that caused irreversible fluidization upon osmotic shock. The terminal component thus creates a fluidized area in the erythrocyte membranes through which small ions and molecules may diffuse more easily and the resulting osmotic unbalance may finally cause hemolysis.     

Mycophenolic acid as a latent agonist of PPARgamma

Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition of IMPDH, we uncovered a hidden biological property of MPA as an agonist of peroxisome proliferator activated receptor gamma (PPARgamma).     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.