Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendai virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nucelar protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100 - 800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenicviral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

Intracellular Lipase Formation by Washed Mycelium Biochemical Studies on Sclerotinia Libertiana. Part 13

Intracellular lipase of the fungus Sclerotina Libertiana Fcl. could be formed powerfully by washed mycelium during shaking in a plain buffer solution, just as well as in the case of shaking culture. Experiments showed revealed it to be favourable to set the mycelium in the experiment harvested at the end of its stationary phase of growth, and that the addition of various respiratory carbon sources had inhibiting effects, while several surface active agents and some enzyme preparations accelerating effects on the lipase formation. Also, the quality and the quantity of consumed cell-materials in the shaking experiment were investigated in relation to lipase formation.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

Psychometric Properties of the Japanese Version of the STarT Back Tool in Patients with Low Back Pain

Background and Objective

The STarT Back Tool uses prognostic indicators to classify patients with low back pain into three risk groups to guide early secondary prevention in primary care. The present study aimed to evaluate the psychometric properties of the Japanese version of the tool (STarT-J).

Methods

An online survey was conducted among Japanese patients with low back pain aged 20–64 years. Reliability was assessed by examining the internal consistency of the overall and psychosocial subscales using Cronbach’s alpha coefficients. Spearman’s correlation coefficients were used to evaluate the concurrent validity between the STarT-J total score/psychosocial subscore and standard reference questionnaires. Discriminant validity was evaluated by calculating the area under the curves (AUCs) for the total and psychosocial subscale scores against standard reference cases. Known-groups validity was assessed by examining the relationship between low back pain-related disability and STarT-J scores.

Results

The analysis included data for 2000 Japanese patients with low back pain; the mean (standard deviation [SD]) age was 47.7 (9.3) years, and 54.1% were male. The mean (SD) STarT-J score was 2.2 (2.1). The Cronbach’s alpha coefficient was 0.75 for the overall scale and 0.66 for the psychosocial subscale. Spearman’s correlation coefficients ranged from 0.30 to 0.59, demonstrating moderate to strong concurrent validity. The AUCs for the total score ranged from 0.65 to 0.83, mostly demonstrating acceptable discriminative ability. For known-groups validity, participants with more somatic symptoms had higher total scores. Those in higher STarT-J risk groups had experienced more low back pain-related absences.

Conclusions

The overall STarT-J scale was internally consistent and had acceptable concurrent, discriminant, and known-groups validity. The STarT-J can be used with Japanese patients with low back pain.     

Dapagliflozin,a Sodium-Glucose Co-Transporter 2 Inhibitor,Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.     

Crystal structure of a hypothetical protein,TTHA0829 from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8, composed of cystathionine-β-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-β-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four β-strands and two α-helices in a βαββαβ motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.     

Septicemia from Lactobacillus rhamnosus GG,from a Probiotic Enriched Yogurt,in a Patient with Autologous Stem Cell Transplantation

Probiotics and Antimicrobial Proteins - Probiotic-rich foods are consumed without much restriction. We report here, a case of septic shock caused by yogurt derived Lactobacillus species in a...     

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.     

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.