Novelty of Sphingolipids in the Central Nervous System Physiology and Disease: Focusing on the Sphingolipid Hypothesis of Neuroinflammation and Neurodegeneration.

For decades, lipids were confined to the field of structural biology and energetics as they were considered only structural constituents of cellular membranes and efficient sources of energy production. However, with advances in our understanding in lipidomics and improvements in the technological approaches, astounding discoveries have been made in exploring the role of lipids as signaling molecules, termed bioactive lipids. Among these bioactive lipids, sphingolipids have emerged as distinctive mediators of various cellular processes, ranging from cell growth and proliferation to cellular apoptosis, executing immune responses to regulating inflammation. Recent studies have made it clear that sphingolipids, their metabolic intermediates (ceramide, sphingosine-1-phosphate, and N-acetyl sphingosine), and enzyme systems (cyclooxygenases, sphingosine kinases, and sphingomyelinase) harbor diverse yet interconnected signaling pathways in the central nervous system (CNS), orchestrate CNS physiological processes, and participate in a plethora of neuroinflammatory and neurodegenerative disorders. Considering the unequivocal importance of sphingolipids in CNS, we review the recent discoveries detailing the major enzymes involved in sphingolipid metabolism (particularly sphingosine kinase 1), novel metabolic intermediates (N-acetyl sphingosine), and their complex interactions in CNS physiology, disruption of their functionality in neurodegenerative disorders, and therapeutic strategies targeting sphingolipids for improved drug approaches.

Transparent and Hard Siloxane Based Hybrid UV-Curable Coating Materials with Amphiphobic Properties.

In this study, highly transparent siloxane-based hybrid UV-curable coating materials were prepared using (acryloxypropyl)methylsiloxane monomer (APMS), a thiol-ene monomer, with benzoin ethyl ether. For the thiol-ene monomer, either pentaerythritol tetrakis(3-mercaptopropionate) (PETTMP) or trimethylolpropane tris(3-mercaptopropionate) (TMPTMP) was used. The siloxane-based hybrid coating materials were highly transparent and hard (pencil hardness of 6-7H). The materials were also amphiphobic, with a water static contact angle of 92-100° and an oil contact angle of 46-63°, when prepared with a high siloxane-monomer-to-PETTMP/TMPTMP ratio. In general, both hybrid coating materials exhibited improved oleophobicity, high hardness, and surface smoothness with increasing siloxane content, although the TMPTMP-based hybrid coating films exhibited slightly higher oleophobicity (lower hydrophobicity) and a smoother surface than the PETTMP-based hybrid coating films.

Effect of Electrical Stimulation on Blood Flow Velocity and Vessel Size.

Interferential current electrical stimulation alters blood flow velocity and vessel size. We aimed to investigate the changes in the autonomic nervous system depending on electrical stimulation parameters. Forty-five healthy adult male and female subjects were studied. Bipolar adhesive pad electrodes were used to stimulate the autonomic nervous system at the thoracic vertebrae 1-4 levels for 20 min. Using Doppler ultrasonography, blood flow was measured to determine velocity and vessel size before, immediately after, and 30 min after electrical stimulation. Changes in blood flow velocity were significantly different immediately and 30 min after stimulation. The interaction between intervention periods and groups was significantly different between the exercise and pain stimulation groups immediately after stimulation (p<0.05). The vessel size was significantly different before and 30 min after stimulation (p<0.05). Imbalances in the sympathetic nervous system, which regulates balance throughout the body, may present with various symptoms. Therefore, in the clinical practice, the parameters of electrical stimulation should be selectively applied in accordance with various conditions and changes in form.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cells in scaffold.

Combination of tissue engineering and cell therapy represents a promising approach for bone regeneration. Human mesenchymal stem cells (hMSCs) have properties that include low immunogenicity, high proliferation rate, and multi-differentiation potential; therefore, they are an attractive seeding source for tissue engineering therapy. Here we found that hMSCs with a scaffold did not affect cell viability and osteogenic differentiation. We also investigated regenerative effect of hMSCs with the scaffold in a calvarial bone defect model. Formation of new bone was evaluated by micro-CT, histology and expression of osteogenic markers. The results clearly showed interesting evidence indicating that hMSCs with scaffold increased the formation of new bone and expression of osteogenic markers, compared to the empty and scaffold only groups. Overall, our results suggest that hMSCs with scaffold are suitable for stimulation of intense bone regeneration in critical-sized bone defects.

Granzyme B leakage-induced apoptosis is a crucial mechanism of cell death in nasal-type NK/T-cell lymphoma.

This study aims to investigate the role of granzyme B in the apoptosis of nasal-type NK/T-cell lymphoma. Twenty-four nasal-type NK/T-cell lymphomas were examined by TdT-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) assay and immunohistochemical staining for active caspase 3, poly(ADP-ribose) polymerase (PARP-1/p85)/p85, and Bcl-2. In addition, HANK-1 and NKL cell lines were analyzed using Western blot analysis. Immunoprecipitation was performed to identify the binding of granzyme B and intrinsic serpin proteinase inhibitor 9 (PI-9). To localize granzyme B, immunogold labeling and immunofluorescence staining were performed. The expression level of granzyme B in tumor tissue was correlated with the apoptosis rate (P=0.015), degree of necrosis (P=0.002), and the levels of active caspase 3 (P=0.036) and poly ADP-ribose polymerase (PARP)-1/p85 (P=0.040). The granzyme B-positive HANK-1 cell line showed increased spontaneous cell death compared to the granzyme B-negative NKL cell line. The untreated HANK-1 cells released cytochrome c into the cytosol with cleavage of caspase 3 and PARP-1. Treatment with granzyme B inhibitor and caspase inhibitor decreased the cleavage of PARP-1. By performing immunogold labeling, granzyme B was identified within the cytolytic granules as well as in the cytosol. Confocal microscopy and immunoprecipitation assays confirmed the colocalization of PI-9 and granzyme B, which formed an SDS-resistant complex. These results suggested that granzyme B leakage induces cell death in NK/T-cell lymphomas via both caspase-dependent and -independent mechanisms, and this leads to the extensive necrosis that is commonly seen in NK/T-cell lymphoma.

Polysaccharides isolated from Phellinus gilvus enhances dermal wound healing in streptozotocin-induced diabetic rats.

Dermal wound healing is a complex process that involved inflammation leading to re-epithelialization, granulation tissue, and tissue remodeling. Previous studies from our laboratory have shown that polysaccharides isolated from fungus, Phellinus gilvus (PG) have various anti-inflammatory activities. In present study, we have assessed the effect of polysaccharides from PG on the dermal wound healing of polysaccharides from PG in streptozotocin-induced diabetic rat model. Six of 6-mm circular wounds were created with biopsy punch on the 4th day after induction of diabetes. After 24 hours, each test substance was applied to the wound twice a day for next 5 days. Circular wounds treated with PG showed significantly reduced wound contraction and complete reepithelialization, as compared to wounds of non-treated (p<0.05). These results show that polysaccharides isolated from PG enhanced wound repair in diabetic impaired healing, and could be developed as a wound healing agent in such clinical settings.

Comparison of intraperitoneal anti-adhesive polysaccharides derived from Phellinus mushrooms in a rat peritonitis model.

AIM: To assess the adhesion- and abscess-reducing capacities of various concentrations of polysaccharides derived from fungus, Phellinus gilvus (PG) or Phellinus linteus (PL) in a rat peritonitis model. METHODS: In 96 SD rats, experimental peritonitis was induced using the cecal ligation and puncture model (CLP). Rats were randomly assigned to 8 groups; Ringer's lactate solution (RL group), hyaluronic acid (HA group), 0.025%, 0.25%, and 0.5% polysaccharides from PG (PG0.025, 0.25, and 0.5 groups), and PL (PL0.025, 0.25, and 0.5 groups). Adhesions and abscesses were noted at 7 d after CLP. RT-PCR assay was performed to assess the cecal tissue. RESULTS: Adhesion formation was significantly reduced in PG0.25, 0.5, PL0.25, 0.5, and HA groups (2.5+/-0.7, 2.4+/-0.7, 3.8+/-1.0, 3.6+/-0.8, and 2.7+/-1.1, P<0.05). The incidence of abscesses was significantly reduced in all treated groups compared to RL group (58%, P<0.05). The urokinase-type plasminogen activator (uPA) gene expression was greatly up-regulated by increasing the concentration of polysaccharides. The urokinase-type plasminogen activator receptor (uPAR) and tumor necrosis factor (TNF)-alpha mRNA were highly expressed in PG0.25, 0.5, PL0.25, and 0.5 groups. CONCLUSION: We concluded that 0.5% polysaccharide derived from PG and PL was the optimal concentration in preventing adhesion and abscess formation and may act by modulating activity of uPA and TNF-alpha in a rat peritonitis model.

Inhibitory effects of polysaccharides isolated from Phellinus gilvus on benzo(a)pyrene-induced forestomach carcinogenesis in mice.

AIM: Although polysaccharides from Phellinus mushrooms are a well-known material with anti-tumor properties, there is no information about the effect of polysaccharides from Phellinus gilvus (PG) on tumor. The modulating effect of polysaccharides isolated from PG on the benzo(a)pyrene (BaP)-induced forestomach carcinogenesis in ICR female mice was investigated in this study. METHODS: A forestomach carcinogenesis model was established in 40 ICR female mice receiving oral administration of BaP for 4 wk. The mice were randomly assigned to 4 groups (10 each). The mice in each group were treated with sterile water or PG for 4 and 8 wk (SW4, PGW4, SW8, and PGW8 groups). Eight or 12 wk after the first dose of BaP, forestomachs were removed for histopathological and RT-PCR analysis. RESULTS: In histopathological changes and RT-PCR analysis, sterile water-treated mice showed significant hyperplasia of the gastric mucosa with a significantly increased expression of mutant p53 mRNA compared to mice treated with PG for 8 wk. CONCLUSION: Polysaccharides isolated from PG may inhibit BaP-induced forestomach carcinogenesis in mice bydown-regulating mutant p53 expression.

Polysaccharides isolated from Phellinus gilvus inhibit melanoma growth in mice.

There is no information about the effect of polysaccharides from fungus, Phellinus gilvus (PG) on melanoma. The effect of PG on the proliferation and apoptosis of the B16F10 melanoma cell line was determined by a sulforhodamine B (SRB) and a sandwich enzyme-linked immunosorbent assay. The in vivo effect of PG on B16F10 melanoma cells allografted in athymic nude mice was investigated. PG decreased cell proliferation and increased cell apoptosis in a dose dependent manner in vitro. Also, PG significantly inhibits melanoma growth in mice. The PG anti-tumor effect in vivo was associated with a significant increase in the melanoma apoptosis rate. These findings support PG as a therapeutic agent against melanoma.

The effect of polysaccharides and carboxymethylcellulose combination to prevent intraperitoneal adhesion and abscess formation in a rat peritonitis model.

Polysaccharides isolated from fungi, Phellinus spp. is well-known material with anti-tumor and anti-inflammatory properties. We have assessed the adhesion- and abscess-reducing capacity of carboxymethylcellulose (CMC) and polysaccharides from Phellinus spp. combination in a rat peritonitis model. In 72 Sprague-Dawley rats, experimental peritonitis was induced by means of the cecal ligation and puncture model (CLP). After 24 hr, the abdomen was reopened and the ligated cecum was resected. Peritoneal fluid samples were taken for microbiological examination. Rats were randomly assigned to 6 groups: ringer lactate solution (RL group), polysaccharides from Phellinus gilvus (PG group) and Phellinus linteus (PL group), carboxymethylcellulose (CMC group), and their combinations (PG+CMC and PL+CMC groups). Adhesions and abscesses were noted at day 7 after CLP. RT-PCR assay for urokinase-type plasminogen activator (uPA), its cellular receptor (uPAR), and tumor necrosis factor (TNF)-alpha was performed to assess the cecal tissue. Microbiological examination showed polymicrobial bacterial peritonitis. Adhesion formation was significantly reduced in PG+CMC and PL+CMC groups (P<0.05). The incidence of abscesses was reduced in all treated groups except the RL group (P<0.05). uPA, uPAR, and TNF-alpha mRNA were highly expressed in the PG+CMC and PL+CMC groups, as compared to the RL group. We concluded that the combination of polysaccharides and CMC had significant adhesion- and abscess-reducing effects compared with their single treatment and the effects may act by modifying the fibrinolytic capacity of uPA, uPAR and TNF-alpha produced from activated macrophages in a rat peritonitis model.

Ex vivo gene therapy using bone marrow-derived cells: combined effects of intracerebral and intravenous transplantation in a mouse model of Niemann-Pick disease.

Normal murine bone marrow cells were transduced with a retroviral vector to overexpress and release human acid sphingomyelinase (ASM). The transduced cells were then transplanted intravenously into 3-day-old, irradiated ASM-deficient mice, a model of human Niemann-Pick disease (NPD). At 4 weeks, engrafted mice received intracerebral injections of mesenchymal stem cells obtained from the original, transduced bone marrow. By 16 weeks, most of the treated NPD mice had near-normal levels of ASM activity in their tissues, including the brain; dramatically improved histology; and marked reductions in sphingomyelin. Cerebellar function also was normal, and the number of Purkinje cells was > 80% of normal. Remarkably, in certain regions of the cerebellum many of the surviving Purkinje cells expressed human ASM RNA, suggesting that either they were donor-derived or that the transplanted bone marrow cells had fused with existing Purkinje cells. However, despite these positive results, by 24 weeks the ASM activities were dramatically reduced and cerebellar function began to decline, coincident with the detection of anti-human ASM antibodies in the plasma. We conclude that this gene therapy procedure might be useful in Type A NPD and other neurological lysosomal storage disorders, particularly since it is an approach that could be beneficial for both the neurological and the visceral organ features of these diseases.

Polymorphisms and the differential antiviral activity of the chicken Mx gene.

The nucleotide sequence of chicken Mx cDNA was reported earlier using the White Leghorn breed in Germany, but it showed no enhanced resistance to viruses. In this study, the nucleotide sequences of chicken Mx cDNA were determined in many breeds. A total of 25 nucleotide substitutions, of which 14 were deduced to cause amino acid exchanges, were detected, suggesting that the chicken Mx gene is very polymorphic. Transfected cell clones expressing chicken Mx mRNA were established after the Mx cDNA was constructed with an expression vector and introduced into mouse 3T3 cells, and the Mx genes from some breeds were demonstrated to confer positive antiviral responses to influenza virus and vesicular stomatitis virus. On the basis of the comparison among the antiviral activities associated with many Mx variations, a specific amino acid substitution at position 631 (Ser to Asn) was considered to determine the antivirally positive or negative Mx gene. Thus, a single amino acid substitution influences the antiviral activity of Mx in domesticated chickens.