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Wild anguillid eel larvae inhabit the ocean during their early life stages and never experience low-salinity water until the glass eel stage. The larvae show less mortality in half-diluted seawater than in full-strength seawater in captivity; however, physiological influences of environmental salinity on eel larvae have not been clarified. In this study, we compared the distributional and functional features of ionocytes between Japanese eel larvae acclimated to half-diluted and full-strength seawater. The mean tissue fluid osmolality in larvae acclimated to half-diluted seawater (300 mOsm/kg H2O) was slightly lower than in those (344 mOsm/kg H2O) acclimated to full-strength seawater. The density and opening size of ionocytes in the skin were not significantly different between the two salinities. Na+/K+-ATPase-immunoreactive ionocytes showed Na+/H+ exchanger-3 (NHE3) and cystic fibrosis transmembrane conductance regulator (CFTR) immunoreactions in their apical region and Na+/K+/2Cl- cotransporter-1 (NKCC1) immunoreaction in their basolateral region, suggesting that the skin ionocytes are involved in salt secretion in both salinities. In transmission electron microscopic observation, the ionocytes of larvae in full-strength seawater were characterized by the electron-dense cytoplasm, expanded tubular system and well-developed mitochondria, compared with those in half-diluted seawater, suggesting that the salt-secreting function was more activated in full-strength seawater than in half-diluted seawater. These results suggest that the energy metabolism cost of ion regulation could be lower in the intermediate salinity environment, which is closer to their osmolality than full-strength seawater. Hence, it is hypothesized that the saving of energy required for osmoregulation in half-diluted seawater could be favorable to better survival and growth of artificial eel larvae.

The centrosome is the major microtubule-organizing center and plays important roles in intracellular transport, cellular morphology, and motility. In mitotic cells, centrosomes function as spindle poles to pull a set of chromosomes into daughter cells. In quiescent cells, primary cilia are originated from the centrosomes. Given its involvement in various cellular processes, it is little surprising that the organelle would also participate in apoptotic events. However, it remains elusive how the centrosome changes in structure and organization during apoptosis. Apoptosis, a programmed cell death, is required for homeostatic tissue maintenance, embryonic development, stress responses, etc. Activation of caspases generates a cascade of apoptotic pathways, explaining much of what happens during apoptosis. Here, we report the proteolytic cleavage of selected centrosomal proteins in apoptotic cells. SAS-6, a cartwheel component of centrioles, was specifically cleaved at the border of the coiled-coil domain and the disordered C-terminus. Pericentrin, a scaffold of pericentriolar material, was also cleaved during apoptosis. These cleavages were efficiently blocked by the caspase inhibitors. We propose that the caspase-dependent proteolysis of the centrosomal proteins may destabilize the configuration of a centrosome. Loss of centrosomes may be required for the formation of apoptotic microtubule networks, which are essential for apoptotic fragmentation. This work demonstrates the first centrosomal targets by caspases during apoptosis.

The anguillid eels are catadromous fishes that migrate between marine and freshwater habitats. The long migration of eel larvae, called leptocephali, as long as thousands of kilometers in the ocean is important to determine their recruitment successes. The leptocephali in the ocean have a pelagic lifestyle totally different from the benthic one of glass eels and yellow eels in rivers. It is known that eel leptocephali have ionocytes on the body surface that may maintain ionic and osmotic status in the internal environment; however, detailed morphology and function of ionocytes in leptocephali are still unknown. In the present study, we aimed 1) to clarify the morphological features of the epidermis in Japanese eel Anguilla japonica leptocephali cultured in hyper-osmotic condition, and 2) to examine the ion-transporting functions of ionocytes of both leptocephali and yellow eels. Na+/K+-ATPase-immunoreactive ionocytes were distributed all over the body surface of leptocephali. Ionocytes were in contact with external environments through their apical membrane, which was located at the boundary of pavement cells. Na+/K+-ATPase-immunopositive cells were not observed in the skin of seawater-acclimated yellow eels. In ionocytes of the larval skin, the apical membrane appeared as a slightly projecting disk with a microvilli-like structure. Meanwhile, the apical membrane of gill ionocytes of yellow eels formed a concave surface. In ionocytes of leptocephali, mitochondria were enlarged and the tubular system was well developed, as compared with those of the gill of yellow eels. Ionocytes of leptocephali showed CFTR immunoreaction in their apical region and NKCC1 immunoreaction in their basolateral region, suggesting that the skin ionocytes are involved in salt secretion. These results support the notion that Japanese eel maintain their ion balance through skin ionocytes during early life stages, and that the skin ionocytes of leptocephali disappear in yellow eel stages after the formation of functional gills and gill ionocytes.

A method for analysis of five artificial sweetners (sodium saccharin, aspartame, acesulfame-K, sucralose, cyclamate) in beverage samples was developed using high-performance liquid chromatography/triple qua- drupole mass spectrometry (HPLC/MS/MS). The method uses a single-step dilution for sample preperation. Sepera- tion was achieved on a C18 column (2.1 × 150 mm, 3.5 µm) with A- 2% methanol (1 mM ammonium acetate), B-95% methanol (1 mM ammonium acetate) as mobile phase with gradient mode. The quantitation of target compounds was performed by external calibration in selected reaction monitorning (SRM) mode. The coefficient of determination of calibration curve for sodium saccharin, aspartame, acesulfame-K, sucralose and cyclamate were 0.9957, 0.9991, 0.9943, 0.9982 and 0.9948, respectively. The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.001~0.022 mg/L and 0.004~0.073 mg/L, repectively. Recoveries for beverage samples were in the range of 92.76~113.50% with RSD < 10.91%. The method has applied to the determination of the five sweetners in 102 bev- erage samples. Three artificial sweetners-aspartame, acesulfame-K, sucralose were detected from 42 samples. Sodium saccharin and cyclamate were not detected in all samples.

Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.

Objective: This research proposes a methodology, which validates a grasp of the customers' emotions in the service design area. Background: As the era of service design has taken its approach, the need for a deliberate design that would reflect the customer's experience had emerged in the area of service. Therefore, a variety of methodologies has been adopted in the field of service design with the purpose of discovery of the customers' needs. Even though the importance of an emotion-sentient research of a service experience increases, its research progress remains to be inadequate in comparison to all the other areas. Method: Having had taken some resources from the emotional studies under other areas of expertise as a base, the concept of volatility of emotions has been introduced as the core element of this research, further followed by an elaboration of its special characteristics. The observation technique under Stakeholder's system: SOMC(Simultaneous Observation Method based on Cooperation) has been proposed in this study as it presents an effective way to grasp the concept of volatile emotions in contrast to the previously existent types of methodologies. Results: The SOMC rather supplements the existing research methods than substitutes the previous ones. In other words, although the existing research system allowed emotion detection, it was difficult to capture the change of momentary and fickle emotions. On the opposite, the SOMC provides a condition allowing a sufficient grasp of the customer's emotions and facilitates emotional capture. Conclusion: For that reason, it is hoped that this piece of research represents a valuable and effective approach in terms of grasping the true needs of the customers on the emotional level, which will in its turn contribute to the improvement of the service quality in the midst of a complicated service condition. Application: Moreover, the purpose of this research is that in its outcome it may serve as a sufficient contribution to the area of emotional studies within the field of service design.

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of D: -glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for D: -glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30 degrees C and 4 degrees C, respectively. The K (m) and V (max) values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 1mole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His(6)-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28 degrees C, starting induction with 0.735% xylose when A (600) was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.

Morphological changes in gill mitochondria-rich (MR) cells were examined in cultured Japanese eel acclimated to deionized freshwater (DFW), freshwater (FW), 30%-diluted seawater (DSW), and seawater (SW). The gill Na+/K+-ATPase activity was higher in SW-acclimated eel than in those acclimated to DFW, FW, and DSW. Immunocytochemical observations revealed that MR cells in the gill filaments were most developed in SW, whereas MR cells in the lamellae were preferentially observed in DFW, suggesting that filament and lamellar MR cells are responsible for ion secretion and absorption, respectively. In scanning electron microscopic observations, the apical membrane of lamellar MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface. In contrast, the apical membrane of filament MR cells showed a slightly concave surface. Whole-mount immunocytochemistry revealed that most MR cells showed cystic fibrosis transmembrane conductance regulator immunoreaction in their apical region in fish in DSW and SW, but not in those in DFW and FW, indicating that MR cells developed in DSW and SW function as an ion-secreting site. In addition to MR cells, distinct Na+/K+-ATPase immunoreaction was observed in the outermost layer of gill epithelia, suggesting that pavement cells are an additional site of ion uptake in the gills.

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae involves the formation of various by-products, which are synthesized through the oxidative pathway. To eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by constructing two mutant strains. In one of the mutant strains, the structural genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were deleted from the chromosomal DNA, whereas in the second mutant strain dhaR, which is a putative transcription factor that activates, gene expression was deleted from the chromosomal DNA. In the resultant mutant strains lacking the dhaT gene encoding 1,3-PD oxidoreductase, which was simultaneously deleted while replacing the native promoter with the lacZ promoter, the by-product formation except for acetate was eliminated, but it still produced 1,3-PD at a lower level, which might be due to a putative oxidoreductase that catalyzes the production of 1,3-PD. The recombinant strains in which the reductive pathway was recovered produced slightly lower amount of 1,3-PD as compared to the parent strain, which might be due to the reduced activity of DhaB caused by the substitution of the promoter. However, the production yield was higher in the recombinant strain (0.57 mol mol(-1)) than the wild type Cu strain (0.47 mol mol(-1)).

The linear isomalto-oligosaccharides (IMO) with DP2–DP10 were produced by one-step process using engineered fusion enzyme (DXSR) of endo-dextranase and only α-(1–6) glucan synthesizing dextransucrase. The fusion enzyme was successfully expressed in Escherichia coli and characterized. Compared to individual enzymes, DXSR had 150% increased endo-dextranase activity and 98% decreased dextransucrase activity. The partially purified DXSR displayed molecular mass of 240 kDa as analyzed by SDS–PAGE. It showed both enzyme activities on analysis by zymogram. The thermal- and pH-stability of DXSR was around 28 °C and pH at 5.0–6.4, respectively. IMOs production by DXSR was increased by the addition of metal ions such as Fe 2+ , Li + , K + and Ni 2+ , but the enzyme was strongly inhibited by Hg 2+ and Ag + . DXSR produced linear IMO with DP2–DP10 using sucrose as a sole substrate. The molecular weight and amount of IMO could be controlled by the sucrose concentration. DXSR gave 30-fold higher production of IMO than that of an equal activity mixture of the two enzymes such as dextranase and dextransucrase.

The ultra precision technology will take a key role in future manufacturing systems in gold wire bonding capillary for semiconductor package technologies industries, so that much more efforts are needed to meet the future demands of semiconductor industries. Due to their high modulus and hardness, low density and resistance to high temperature and corrosive environment, there is a great interest in using ceramics in demanding structural applications such as bonding capillary, optical lens body, and LC (liquid crystal) nozzle, where their use would result in long life, operation at high temperatures and weight saving. The microstructure of ZrO 2 toughened Al 2 O 3 ceramics and Al 2 O 3 –Cr 2 O 3 ceramics (ruby) were carefully controlled so as to obtain dense and fine-grained ceramics, thereby improving the properties and reliability of the ceramics for capillary applications in semiconductor bonding technology. The composites were produced via ceramic injection molding technology, followed by sintering/hot isostatic pressing (HIP). Room temperature strength, hardness, Young's modulus, thermal expansion coefficient and toughness were determined, as well as surface strengthening induced by the fine-grained homogenous microstructure and the thermal treatment. The changes in microstructure grain size, sintering condition and HIP treatment were found to be correlated.

We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSHβ/α and LHβ/α, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7–9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSHα-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSHβ-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSHβ/α and LHβ/α produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSHβ/α was detected. The activity of rec-LHβ/α was found to be increased in a dose-dependent manner for eel oocyte maturation.

The microstructure of ZrO₂ toughened Al₂O₃ ceramics was carefully controlled so as to obtain dense and fine-grained ceramics, thereby improving the properties and reliability of the ceramics for capillary applications in semiconductor bonding technology. Al₂O₃-ZrO₂(Y₂O₃) composite was produced via Ceramic Injection Molding (CIM) technology, followed by Sinter-HIP process. Room temperature strength, hardness, Young’s modulus, thermal expansion coefficient and toughness were determined, as well as surface strengthening induced by the fine grained homogenous microstructure and the thermal treatment. The changes in alumina/zirconia grain size, sintering condition and HIP treatment were found to be correlated.

We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ∼500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N- glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.

Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU) 8 has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative π-facial hydrogen bond between the 2′-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the π-facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.

The effects of Cr₂O₃ on the microstructural evolution and mechanical properties of Al₂O₃ polycrystalline were investigated. The microstructure of Al₂O₃-Cr₂O₃ composites (ruby) was carefully controlled in order to obtain dense and fine?grained ceramics, thereby improving their properties and reliability with respect to numerous applications related to semiconductor bonding technology. Ruby composites were produced by Ceramic Injection Molding (CIM) technology. Room temperature strength, hardness, Young’s modulus and toughness were determined, as well as surface strengthening induced by thermal treatment and production of a fine-grained homogenous microstructure.

We examined morphological changes and molecular mechanisms of ion regulation in mitochondrion-rich (MR) cells of Japanese eel acclimated to different environmental salinities. Electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface in eel acclimated to freshwater (FW). In seawater (SW)-acclimated eel, in contrast, the apical membrane of MR cells showed a slightly concave surface without a mesh-like structure. The mRNA expression of Na+/H+ exchanger-3 (NHE3) in deionized FW and normal SW was higher than that in normal FW and 30%-diluted SW. Expression of Na+/K+/2Cl− cotransporter-1a (NKCC1a) became higher with increasing environmental salinity. Immunofluorescence staining showed that the apical NHE3 immunoreaction was stronger in deionized FW and normal SW than in the other groups. Basolateral NKCC1 immunoreaction was most intense in normal SW. These results indicate that apical NHE3 is involved in ion uptake in fish acclimated to hypotonic environments, and that basolateral NKCC1 is important for acclimation to hypertonic environments. The relatively high expression of NHE3 in SW further indicates a possible role of NHE3 in acid-base regulation in the gills in SW-acclimated fish.

RNA interference (RNAi) is a process of posttranscriptional gene silencing in plants, insects, and animals (4, 7, 13, 14). In vivo, RNAi is initiated by an endonuclease, DICER, that cleaves long, double-stranded RNA into 21- to 25-bp small interfering RNAs (siRNAs) (2, 3, 6, 15). siRNAs are incorporated into a protein complex, the RNAi silencing complex, that recognizes and cleaves target mRNAs (12). Inhibition of viral infection and modulation of viral replication by siRNA have been demonstrated in human immunodeficiency virus and poliovirus in human cells (5, 8, 10, 11). Hepatitis C virus (HCV) is a positive-stranded RNA virus that has infected some 170 million worldwide. Most HCV-infected individuals develop a chronic infection that can lead to liver disease, including cirrhosis and hepatocellular carcinoma (1). To test whether siRNA inhibits HCV, we made several 21-bp siRNA duplexes that were directed against different regions of the 5′ untranslated region (UTR) of the HCV genome. We transfected these siRNAs into 5-2 cells, a Huh-7 cell line that harbors autonomously replicating subgenomic HCV RNA (9) (Fig. ​(Fig.1A).1A). The subgenomic replicon contains the firefly luciferase gene; therefore, the effect of siRNA on HCV replication was measured through the luciferase assay (9). Compared with those of mock-transfected controls, luciferase activities measured 48 h after transfection of cells with HCV siRNA 5U5 (Fig. ​(Fig.1B)1B) were reduced by up to 85% in a dose-responsive manner (Fig. ​(Fig.2A).2A). This inhibition was not seen in cells that were transfected with the control siRNA SIN, which was targeted to Sindbis virus. Additional control siRNA duplexes, GL2 and GL3, further demonstrated the specificity of the siRNA response. Luciferase activity was reduced by 90% in cells transfected with GL2, which is homologous to the fruit fly luciferase gene. GL3, which contains three nucleotide mismatches to the gene, was unable to inhibit luciferase activity effectively (Fig. ​(Fig.2A).2A). We also tested possible cellular toxicity involved in the siRNA transfections. Measurement of ATP levels within cells showed no viable change in siRNA-transfected cells (Fig. ​(Fig.2B).2B). Finally, we tested the effect of 5U5 on HCV RNA replication in 5-2 cells by a quantitative TaqMan assay. This assay showed that 5U5 could reduce the steady-state level of the HCV RNA in a pattern similar to that obtained with the luciferase assay (data not shown). FIG. 1. Reporter HCV replicon and siRNA duplexes. (A) The components of HCV subgenomic replicons are the HCV 5′ UTR, Neo (neomycin phosphotransferase gene), Luc (firefly luciferase reporter gene), the encephalomyocarditis virus (EMCV) internal ribosome ... FIG. 2. siRNA-mediated inhibition of HCV replication as measured by luciferase activity. (A) Effects of siRNA dose and sequence on luciferase activity. The indicated amounts of siRNA were transfected into 5-2 cells (5 × 104 in a 24-well plate) with 1 ... Our results show that a 21-bp siRNA is capable of mediating specific cleavage of target HCV RNA in human liver cells, implying that the target cell for HCV infection possesses the necessary functional components (RNAi silencing complex) of RNAi. Our observation, however, does not rule out the possibility that HCV interferes with the ability of DICER to generate siRNAs. Since HCV is very effective at persisting in the infected host, it may have evolved a mechanism(s) by which it evades the normal siRNA pathway. However, introduction of a preformed siRNA duplex would bypass the required DICER maturation in HCV-infected cells, making the standard siRNA strategy attractive. This report is a first step in demonstrating that replicating HCV RNA is amenable to siRNA-mediated degradation in human liver cells. Identification of siRNA with higher efficacy, greater stability, and targeting of siRNA is necessary to develop siRNA technology into an effective anti-HCV therapy.

The microstructure of zirconia (ZrO2) toughened alumina (Al2O3) ceramics was carefully controlled so as to obtain dense and fine–grained ceramics, thereby improving the properties and reliability of the ceramics for capillary applications in semiconductor bonding technology. Al2O3‐ZrO2(Y2O3) composite was produced via Ceramic Injection Molding (CIM) technology, followed by Sinter‐HIP process. Room temperature strength, hardness, Young's modulus, thermal expansion coefficient and toughness were determined, as well as surface strengthening induced by the fine grained homogenous microstructure and the thermal treatment. The changes in alumina/zirconia grain size, sintering condition and HIP treatment were found to be correlated.

A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit.

Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene (m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 degrees C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 micromol min-1 mg-1.

HRC is a SR luminal protein that binds to both triadin and SERCA, and affects Ca2+ cycling in the SR (Arvanitis et al. Am. J. Physiol. Heart. Circ. Physiol. 293: H1581, 2007). In the present study, we attempted to characterize the functions of HRC by AAV-mediated KD using TAC-HF model. We expected that HRC KD could recover cardiac function in failing heart (FH), because HRC KD by itself could enhance Ca2+ uptake and Ca2+ release by increased activities of SERCA2 and RyR2 in HL-1 cells. Unexpectedly, AAV9-mediated HRC KD in TAC-FH showed decreased fractional shortening and increased cardiac fibrosis compared with control TAC-FH. We found that phospho-RyR2, phospho-CaMKII, phospho-p38MAPK and phospho-PLB Thr17 were significantly up-regulated in HRC-KD TAC-FH. The cardiac cell death markers such as LC3A/B and active caspase 9 were also increased, consistent with our results of TUNEL assay. Collectively, the increased cytosolic Ca2+ level could activate CaMKII and hence phosphorylation of p38MAPK causing the enhanced mitochondrial death pathway in TAC-FH. Our results show evidence that down-regulation of HRC is linked to deterioration of cardiomyocytes in the pathological state. (Supported by Korea NRF Grant (2010-0002159), GIST Systems Biology Infrastructure Establishment Grant (2010) and KISTI-KREONET (2010).