Effect of Lactobacillus pentosus S-PT84 ingestion on IFN-α production from plasmacytoid dendritic cells by virus stimulation

We investigated the effect of ingesting Lactobacillus pentosus S-PT84 on the interferon-α (IFN-α) production from splenocytes and plasmacytoid dendritic cells by virus stimulation. IFN-α production by the Lactobacillus pentosus S-PT84 ingestion group was significantly greater under the virus-infected condition than that by the control group. Lactobacillus pentosus S-PT84 could enhance the production of IFN-α which is known as an important cytokine for preventing virus infection. It may therefore become a prophylactic tool against such virus infection.     

Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second β-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the β-anomer of (GlcNAc)?. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid β-chitin, producing only (GlcNAc)?. The AcMNPV chitinase processively hydrolyzes solid β-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.     

Effect of water flow velocity on growth and morphology of cultured <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> sporophytes (Laminariales,Phaeophyceae) in Okirai Bay on the Sanriku coast,Northeast Japan

Undaria pinnatifida sporophytes, originating from the same strain, were cultured at the commercial cultivation site exposed to wave action and the uncultivated site protected from water action of Okirai Bay, Northeast Japan, from January to April 2007; simultaneously, water flow velocity, water temperature, salinity, NO₃ + NO₂, and chlorophyll a were monitored to investigate the effect of water environment on their growth and morphology. Water temperature and salinity fluctuated within the optimal range for their growth whereas water flow velocity at the cultivation site was greatly fast compared with that at the uncultivated site. Successive chlorophyll a increases synchronized with NO₃ + NO₂ decreases were observed only at the uncultivated site for over a month; indicating developments of phytoplankton blooms and their nutrient consumption under the low-flow condition. Meanwhile, blade growth rate of cultured sporophytes was higher at the cultivation site than at the uncultivated site. Their thallus size expressed by six morphological characters (blade length, stipe length, blade wet weight, stipe wet weight, blade width, and undivided blade width) at the cultivation site became large in comparison with that at the uncultivated site. Their three morphological correlations (correlations between blade length and thallus length; blade wet weight and thallus wet weight; and undivided blade width and blade width) differed between the sites. They produced a thick and flat blade at the cultivation site but formed a thin and wrinkled blade at the uncultivated site. These results show the significant impact of water flow velocity on their growth and morphology.     

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.     

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses

Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ～330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ～128 kDa (～32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.     

Connexin32 protects against vascular inflammation by modulating inflammatory cytokine expression by endothelial cells

Gap junctions (GJs) play an important role in vascular function, stability, and homeostasis in endothelial cells (ECs), and GJs are comprised of members of the connexin (Cx) family. GJs of vascular ECs are assembled from Cx37, Cx40, and Cx43, and we showed that ECs also express Cx32. In this study, we investigated a potential role for Cx32 during vascular inflammation. Expression of Cx32 mRNA and protein by human umbilical venous ECs (HUVECs) decreased following treatment with tumor necrosis factor (TNF)-α, but lipopolysaccharide (LPS) and interleukin (IL)-1β did not affect Cx32 expression. Intracellular transfer of an inhibitory anti-Cx32 monoclonal antibody significantly enhanced TNF-α-induced monocyte chemotactic protein (MCP)-1 and IL-6 expression, but overexpression of Cx32 abrogated TNF-α-induced MCP-1 and IL-6 expression. LPS treatment of Cx32 knock-out mice significantly increased the serum concentrations of TNF-α, interferon-γ, IL-6 and MCP-1, compared to wild-type littermate mice. These data suggest that Cx32 protects ECs from inflammation by regulating cytokine expression and plays an important role in the maintenance of vascular function.     

Egg viability of the rotifer Brachionus urceolaris after ingestion by the predatory cladoceran Leptodora kindtii

Egg resistance against the digestive process of a predator is an effective strategy for zooplankton to compensate population loss due to predation. Parthenogenetic eggs of the rotiferan Brachionus urceolaris, which were ingested by the predatory cladoceran Leptodora kindtii, were expelled from the feeding basket of the predator without digestion. We found a negative correlation between the unconsumed ratio of eggs after ingestion and body length of the predator. As high as 75% of the unconsumed eggs successfully hatched and the hatch ratio was independent of body length of L. kindtii. Our results indicate that the rotifer has an effective strategy to maintain its population in the environment with abundant invertebrate predators.     

A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.     

Synthesis of 3- and 21-monosulfates of [2,2,3β,4,4-²H₅]-tetrahydrocorticosteroids in the 5β-series as internal standards for mass spectrometry

The 3- and 21-monosulfates of pentadeuterated 5β-tetrahydrocorticosteroides were synthesized, starting from cortisol and 11-deoxycotisol. The principal reactions used were (1) perdeuteration of the methylene groups adjacent to the 3-oxo group of 17,20:20,21-bismethylendioxy-5β-3-ketosteroids with NaOD in CH₃OD followed by stereoselective reduction with NaBD₄, (2) sulfation of hydroxy groups with sulfur trioxide–trimethylamine complex, and (3) removal of the 17,20:20,21-bismethylendioxy group with hydrogen fluoride. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.