TNF-α Acts as an Immunoregulator in the Mouse Brain by Reducing the Incidence of Severe Disease Following Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis.     

A Novel Prolyl Hydroxylase Inhibitor Protects Against Cell Death After Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.     

Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood–brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.     

Spatial and temporal progress of programmed cell death in the developing starchy endosperm of rice

Programmed cell death (PCD) is the genetically regulated disassembly of cells, and occurs in the endosperm of cereals during seed maturation. Since PCD determines the lifetime of cells, it can affect endosperm growth and, therefore, cereal yield. However, the features and mechanisms of PCD in the developing starchy endosperm in the Poaceae remain unclear. In the present study, we investigated the characteristics of PCD in developing starchy endosperm of rice (Oryza sativa L.) by fluorescence microscopy, focusing on the spatial and temporal progress of PCD-associated responses. Cell death commenced in the central region of starchy endosperm, and then spread to the peripheral region. PCD-associated responses, such as mitochondrial membrane permeabilization and activation of the protease that cleaves the amino acid sequence VEID, showed similar spatial patterns to that of cell death, but preceded cell death. Degradation of nuclear DNA could not be detected in developing starchy endosperm by the TUNEL assay. These results indicated that PCD in developing starchy endosperm of rice proceeds via a highly organized pattern. In addition, these results suggested that PCD in developing starchy endosperm of rice is characterized by the involvement of mitochondrial signaling and the activity of a caspase-like protease that cleaves the VEID sequence.     

RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.     

Active Bacterial Populations and Grazing Impact Revealed by an In Situ Experiment in a Shallow Aquifer

To elucidate bacterial population dynamics in an aquifer, we attempted to reveal the impact of protozoan grazing on bacterial productivity and community structure by an in situ incubation experiment using a diffusion chamber. The abundance and vertical distribution of bacteria and protozoa in the aquifer were revealed using wells that were drilled in a sedimentary rock system in Itako, Ibaraki, Japan. The water column in the wells possessed aerobic and anaerobic layers. Active bacterial populations under the grazing pressure of protozoa were revealed through in situ incubation with grazer eliminating experiment by the filtration. On August 19, 2003, the total number of bacteria (TDC) decreased from 1.5 × 10⁶ cells ml^{? 1} at 2.2 m depth to 3.0 × 10⁵ cells ml^{? 1} at 10 m depth. The relative contribution of the domain Bacteria to TDC ranged between 63% and 84%. Protozoa existed at a density of 4.2 × 10⁴ to 1.9 × 10⁵ cells ml^{? 1} in both aerobic and microaerobic conditions. A grazing elimination experiment in situ for 6 days brought about clearly different bacterial community profiles between the 2.2 m and 10 m samples. The bacterial composition of the initial community was predominantly β- and γ -proteobacteria at 2.2 m, while at 10 m β-, α - and γ -proteobacteria represented 56%, 26% and 13% of the community, respectively. The distribution of bacterial abundance, community composition and growth rates in the subsurface were influenced by grazing as well as by geochemical factors (dissolved oxygen and concentrations of organic carbon, methane and sulfate). Results of the in situ incubation experiment suggested that protozoan grazing contributes significantly to bacterial population dynamics.     

Circular causality in integrative multi-scale systems biology and its interaction with traditional medicine

This paper discusses the concept of circular causality in “biological relativity” (Noble, Interface Focus. 2, 56-64, 2012) in the context of integrative and multi-scale systems approaches to biology. It also discusses the relationship between systems biology and traditional medicine (sometimes called scholarly medical traditions) mainly from East Asia and India. Systems biology helps illuminate circular processes identified in traditional medicine, while the systems concept of attractors in complex systems will also be important in analysing dynamic balance in the body processes that traditional medicine is concerned with. Ways of nudging disordered processes towards good attractors through the use of traditional medicines can lead to the development of new ways not only of curing disease but also of its prevention. Examples are given of cost-effective multi-component remedies that use integrative ideas derived from traditional medicine.     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

Leucine Dehydrogenase from Corynebacterium pseudodiphtheriticum: Purification and Characterization

Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain l-amino acids, l-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for l-leucine, and the k_cat/Km value for l-leucine was higher than that for l-valine. The enzyme required NAD⁺ as a natural coenzyme. The NAD⁺ analogs 3-acetylpyridine-NAD⁺ and deamino-NAD⁺ were much better coenzymes than NAD ⁺. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5′-phosphate. d-Enantiomers of the substrate amino acids competitively inhibited the oxidation of l-valine.