Increased Solubility of High-Molecular-Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of l -[³⁵S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.     

Synthesis and structure based optimization of 2-(4-phenoxybenzoyl)-5-hydroxyindole as a novel CaMKII inhibitor

Based on 2-(4-phenoxybenzoyl)-5-hydroxyindole (2), a novel structural class of CaMKII inhibitors were synthesized and further optimized. The strong acidity of the hydroxyl group and the lipophilic group at the 4 and 6-positions were found to be necessary for strong CaMKII inhibition. Compound 25 was identified as a promising compound with 50-fold more potent inhibitory activity for CaMKII than 2. Compound 25 also showed high selectivity for CaMKII over off-target kinases.     

The Effect of Lipid Peroxide on the Lipid and Carbohydrate Metabolism in Rat Liver

Feeding tests were carried out on rats to clarify the mechanisms of fatty liver formation induced by autoxidized methyl linoleate. Lipid peroxides prepared by autoxidation of highly purified methyl linoleate were given orally to rats. Triglyceride and glycogen contents in liver were determined and enzyme activities including triglyceride synthetase and α-glycerophosphate dehydrogenase were also examined. The following results were obtained. 1. Triglyceride accumulation in rat liver fed autoxidized methyl linoleate was observed. 2. Increase in triglyceride content in rat liver was soon followed by the decrease of hepatic glycogen. 3. When rats were starved prior to introduction of autoxidized methyl linoleate, hepatic triglyceride accumulation did not occur. 4. The activities of α-glycerophosphate dehydrogenase and triglyceride synthetase in liver, and those of glutamic oxalacetic transaminase and leucine aminopeptidase in plasma were practically similar among the rats of test groups fed fresh or autoxidized methyl linoleate and the control fed diet without methyl linoleate. 5. The addition of l-carnitine which is a stimulator of fatty acid oxidation retarded the accumulation of the hepatic triglyceride mentioned above.     

THE BLOCKAGE OF AXOPLASMIC FLOW OF PROTEINS BY COLCHICINE AND CYTOCHALASINS A AND B

Abstract— Colchichine blocks axoplasmic flow of proteins in chicken sciatic nerve. The slow component is more effectively blocked than the fast. The colchicine effect on slow flow is independent of the time delay between colchicine injection and that of the [¹⁴C]-leucine used to measure flow, over a period extending from 2 h after to 9 days before the leucine. It is still effective, but to a lesser degree, after a period of 27 days. There is little effect on the fast component when the colchicine is administered after leucine. When given before leucine the effect is not pronounced up to a time interval of 1 day. Maximum blockage was obtained with longer intervals of up to 27 days. These results are discussed in relation to the involvement of the microtubules in both slow flow and rapid flow.
The effect of both cytochalasins A and B on slow and rapid protein flow has also been studied. Neither drug had any significant effect on slow flow. Cytochalasin A reduced the amount of protein flowing at the rapid rate to a small extent when administered 2 h before [¹⁴C]leucine. Cytochalasin B also caused a similar reduction and this effect was independent of dose over the ranges of doses used. The cytochalasin B diminished the incorporation of amino acid into protein in the spinal cord and it has been concluded that these results are due to a membrane effect which reduces uptake of amino acid rather than a direct effect on neurofilaments in the axons.     

Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-β administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-β alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-β and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-β, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.     

Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.