Competitive solubilization of cholesterol and β-sitosterol with changing biliary lipid compositions in model intestinal solution

We used a model intestinal solution to understand the mechanisms of cholesterol lowering by the addition of plant sterols. The experimental results of the competitive solubilization of cholesterol and β-sitosterol in vitro give useful information about these mechanisms. The states of the model intestinal solution as a solubilizer were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) by changing the number of components, and the bile salt and phosphatidylcholine concentrations. There were aggregates of different sizes: liposomes and mixed micelles depending on their components and concentrations. The maximum solubilization of cholesterol increased from 0.2mM to 1.3mM when adding fatty compounds in the pure bile salts system, which is almost the same as the full components model intestinal solution. Therefore, an excessive intake of fatty compounds may also increase cholesterol absorption in vivo. Even if the components of the model intestinal solution were modified from the standard condition, there were not remarkable differences in the selectivity of cholesterol and β-sitosterol in competitive solubilization. With the addition of β-sitosterol, the maximum solubilization of cholesterol decreases to almost half of that in the system with only cholesterol, except for PC-rich systems. In general, the different structures of aggregates considerably influence the maximum solubilization of sterols but not the selectivity of cholesterol and β-sitosterol in the competitive solubilization. The Gibbs energy change (ΔG°) of the solubilization of β-sitosterol showed a more negative value than cholesterol by -4 to -6kJmol(-1), which indicates that β-sitosterol is energetically favored relative to cholesterol in the model intestinal solution, regardless of the different systems.     

Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelid): proliferation and migration of neoblasts

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E. japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast‐like cells, which are designated as N‐cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling‐experiments, we have shown that neoblasts are slow‐cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N‐cells proliferate more actively than do neoblasts in intact worms. The results of pulse‐chase experiments indicated that neoblast and N‐cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej‐vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E. japonensis.     

Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus

Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.     

Baculovirus GP64-mediated entry into mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.     

Malaria parasites require TLR9 signaling for immune evasion by activating regulatory T cells

Malaria is still a life-threatening infectious disease that continues to produce 2 million deaths annually. Malaria parasites have acquired immune escape mechanisms and prevent the development of sterile immunity. Regulatory T cells (Tregs) have been reported to contribute to immune evasion during malaria in mice and humans, suggesting that activating Tregs is one of the mechanisms by which malaria parasites subvert host immune systems. However, little is known about how these parasites activate Tregs. We herein show that TLR9 signaling to dendritic cells (DCs) is crucial for activation of Tregs. Infection of mice with the rodent malaria parasite Plasmodium yoelii activates Tregs, leading to enhancement of their suppressive function. In vitro activation of Tregs requires the interaction of DCs with parasites in a TLR9-dependent manner. Furthermore, TLR9(-/-) mice are partially resistant to lethal infection, and this is associated with impaired activation of Tregs and subsequent development of effector T cells. Thus, malaria parasites require TLR9 to activate Tregs for immune escape.     

Interleukin-1 stimulates glutamate uptake in glial cells by accelerating membrane trafficking of Na+/K+-ATPase via actin depolymerization

Interleukin-1 (IL-1) is a mediator of brain injury induced by ischemia, trauma, and chronic neurodegenerative disease. IL-1 also has a protective role by preventing neuronal cell death from glutamate neurotoxicity. However, the cellular mechanisms of IL-1 action remain unresolved. In the mammalian retina, glutamate/aspartate transporter (GLAST) is a Na(+)-dependent, major glutamate transporter localized to Müller glial cells, and loss of GLAST leads to glaucomatous retinal degeneration (T. Harada, C. Harada, K. Nakamura, H. A. Quah, A. Okumura, K. Namekata, T. Saeki, M. Aihara, H. Yoshida, A. Mitani, and K. Tanaka, J. Clin. Investig. 117:1763-1770, 2007). We show here that IL-1 increases glutamate uptake in Müller cells by a mechanism that involves increased membrane Na(+)/K(+)-ATPase localization, required for counteracting the Na(+)-glutamate cotransport. IL-1 activated the p38 mitogen-activated protein kinase (MAPK)/capase 11 pathway, which destabilizes the actin cytoskeleton allowing Na(+)/K(+)-ATPase membrane redistribution. Furthermore, pretreatment with IL-1 protected retinal neurons from glutamate neurotoxicity through p38 MAPK signaling. Our observations suggested that IL-1 acts as a potential neuroprotective agent by modulating the functions of the glia-neuron network.     

Study of thermodynamic parameters for solubilization of plant sterol and stanol in bile salt micelles

We investigated the difference between the molecular structures of plant sterols and stanols that affect the solubilization of cholesterol in bile salt micelles (in vitro study). First, the aqueous solubility of beta-sitosterol, beta-sitostanol, and campesterol was determined by considering the specific radioactivity by using a fairly small quantity of each radiolabeled compound. The order of their aqueous solubilities was as follows: cholesterol > campesterol > beta-sitostanol > beta-sitosterol. The maximum solubility of cholesterol and the above mentioned sterol/stanol in sodium taurodeoxycholate and sodium taurocholate solutions (single solubilizate system) was measured. Moreover, the preferential solubilization of cholesterol in bile salt solutions was systematically studied by using different types of plant sterols/stanols. The solubilization results showed that the cholesterol-lowering effect was similar for sterols and stanol. Thermodynamic analysis was applied to these experimental results. The Gibbs energy change (Delta G degrees ) for the solubilization of plant sterols/stanols showed a negative value larger than that for cholesterol.     

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca(2+)-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca(2+). Ca(2+)-dependent p94 mutants were screened using "p94-trapping", which is an application of yeast genetic reporter system called "proteinase-trapping". Several amino acids were revealed as critical for apparent Ca(2+)-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.