Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.     

Nardilysin enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of tumor necrosis factor-alpha-converting enzyme

Like other members of the epidermal growth factor family, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a transmembrane protein that can be shed enzymatically to release a soluble growth factor. Ectodomain shedding is essential to the biological functions of HB-EGF and is strictly regulated. However, the mechanism that induces the shedding remains unclear. We have recently identified nardilysin (N-arginine dibasic convertase (NRDc)), a metalloendopeptidase of the M16 family, as a protein that specifically binds HB-EGF (Nishi, E., Prat, A., Hospital, V., Elenius, K., and Klagsbrun, M. (2001) EMBO J. 20, 3342-3350). Here, we show that NRDc enhances ectodomain shedding of HB-EGF. When expressed in cells, NRDc enhanced the shedding in cooperation with tumor necrosis factor-alpha-converting enzyme (TACE; ADAM17). NRDc formed a complex with TACE, a process promoted by phorbol esters, general activators of ectodomain shedding. NRDc enhanced TACE-induced HB-EGF cleavage in a peptide cleavage assay, indicating that the interaction with NRDc potentiates the catalytic activity of TACE. The metalloendopeptidase activity of NRDc was not required for the enhancement of HB-EGF shedding. Notably, a reduction in the expression of NRDc caused by RNA interference was accompanied by a decrease in ectodomain shedding of HB-EGF. These results indicate the essential role of NRDc in HB-EGF ectodomain shedding and reveal how the shedding is regulated by the modulation of sheddase activity.     

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

The homolog of Ciboulot in the termite (<Emphasis Type="Italic">Hodotermopsis sjostedti</Emphasis>): a multimeric β-thymosin involved in soldier-specific morphogenesis

Background  

Caste differentiation in social insects is a type of polyphenism that enables division of labor among members of a colony. This elaborate social integration has attracted broad interest, although little is known about its regulatory mechanisms, especially in Isoptera (termites). In this study, we analyzed soldier differentiation in the damp-wood termite Hodotermopsis sjostedti, focusing on a possible effector gene for caste development. The gene for an actin-binding protein, HsjCib, which shows a high level of expression in developing mandibles during soldier differentiation, is characterized in detail.     

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.     

Heritability of male mandible length in the stag beetle Cyclommatus metallifer

Numerous coleopteran species express male‐specific “weapon traits” that often show size variations among males, even within a single population. Many empirical studies have demonstrated that environmental conditions during development affect absolute weapon size. However, relatively few studies in horned beetles support the hypothesis that the relationship between weapon size and body size, also referred to as a “scaling relationship” or “static allometry”, is largely determined by genetic factors. In this study, the heritability of absolute mandible length and static allometry between mandible length and body size were estimated in the stag beetle Cyclommatus metallifer. While no significant heritable variation was observed in absolute mandible length, high heritability (h² = 0.57 ± 0.25) was detected in the static allometry between mandible length and body size. This is the first report on the genetic effect on male mandible size in Lucanidae, suggesting that absolute mandible size is largely determined by environmental conditions while the static allometry between weapon size and body size is primarily determined by genetic factors.     

Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T_H2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺ iNKT cells are present in the thymic CD44^+/− NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻ iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺ iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺ iNKT cells produce T_H2 (IL-13), T_H9 (IL-9 and IL-10), and T_H17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺ iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T_H17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺ iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺ iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.