The effect of cannabichromene on adult neural stem/progenitor cells

Apart from the psychotropic compound Δ⁹-tetrahydrocannabinol (THC), evidence suggests that other non-psychotropic phytocannabinoids are also of potential clinical use. This study aimed at elucidating the effect of major non-THC phytocannabinoids on the fate of adult neural stem progenitor cells (NSPCs), which are an essential component of brain function in health as well as in pathology. We tested three compounds: cannabidiol, cannabigerol, and cannabichromene (CBC), and found that CBC has a positive effect on the viability of mouse NSPCs during differentiation in vitro. The expression of NSPC and astrocyte markers nestin and Glial fibrillary acidic protein (GFAP), respectively, was up- and down-regulated, respectively. CBC stimulated ERK1/2 phosphorylation; however, this effect had a slower onset in comparison to typical MAPK stimulation. A MEK inhibitor, U0126, antagonized the up-regulation of nestin but not the down-regulation of GFAP. Based on a previous report, we studied the potential involvement of the adenosine A1 receptor in the effect of CBC on these cells and found that the selective adenosine A1 receptor antagonist, DPCPX, counteracted both ERK1/2 phosphorylation and up-regulation of nestin by CBC, indicating that also adenosine is involved in these effects of CBC, but possibly not in CBC inhibitory effect on GFAP expression. Next, we measured ATP levels as an equilibrium marker of adenosine and found higher ATP levels during differentiation of NSPCs in the presence of CBC. Taken together, our results suggest that CBC raises the viability of NSPCs while inhibiting their differentiation into astroglia, possibly through up-regulation of ATP and adenosine signalling.     

The use of BRM-activated killer cells in adoptive immunotherapy: A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN- and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated T cells producing IFN- were assayed as an indication marker of BAK therapy. The normal range of IFN- producing T cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN- producing T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood T cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN- producing T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial T cell counts, a low frequency of these cells may contraindicate BAK therapy.     

Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.     

SIRPα interacts with nephrin at the podocyte slit diaphragm

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in?vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury. Structured digital abstract ? Sirp-alpha?physically interacts?with?Nephrin?by?anti bait coimmunoprecipitation?(View interaction) ? Sirp-alpha?physically interacts?with?Nephrin?by?anti tag coimmunoprecipitation?(View interaction).     

Degradation of 17β-Estradiol by a Gram-Negative Bacterium Isolated from Activated Sludge in a Sewage Treatment Plant in Tokyo, Japan

A 17β-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new Novosphingobium species. Gas chromatography-mass spectrometry and ¹H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.     

A structural study of the asparagine-linked oligosaccharide moiety of duck ovomucoid

Asparagine-linked oligosaccharides of duck ovomucoid were released quantitatively from the protein by digestion with glycoamidase A (from almond), the reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated using two different types of high performance liquid chromatography (HPLC) on a reversed phase column and an amide adsorption column. More than sixteen different oligosaccharides were separated and the structures were characterized by a combination of the 2-dimensional sugar mapping technique using HPLC, exoglycosidase digestion, and proton nuclear magnetic resonance measurements (1- and 2-dimensional). Furthermore, the HPLC profile of duck ovomucoid oligosaccharides was compared with previously reported profiles obtained from quail and chicken ovomucoids.Abbreviations COSY chemical shift-correlated spectroscopy - DQF-COSY double quantum filtered COSY - DSS sodium, 4,4-dimethyl-4-silapentane 1-sulfonate - Gal D-galactose - GlcNAc or GN N-acetyl-D-glucosamine - HOHAHA homonuclear Hartmann-Hahn spectroscopy - Man or M D-mannose - NOE nuclear Overhauser enhancement - ODS octadecylsilyl - PA pyridylamino - ROESY rotating frame nuclear Overhauser effect spectroscopy - SDS/PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis