Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.     

Multiple fates of non‐mature lumenal proteins in thylakoids

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N–terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid‐transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling‐lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1‐null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid‐transfer signal removal on protein targeting and accumulation by examining the localization of non‐mature lumenal proteins in the Arabidopsis plsp1‐null mutant and performing a protein import assay using pea chloroplasts. In plsp1‐null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non‐mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid‐transfer signal removal did not disrupt cpSEC‐ and cpTAT‐dependent translocation, but prevented release of proteins from the membrane. Interestingly, non‐mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440‐kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1‐null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.     

Synthesis and biological evaluation of novel tryptoline derivatives as indoleamine 2,3-dioxygenase (IDO) inhibitors

Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer’s disease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and evaluated for their inhibitory activity against IDO. Substituted tryptoline derivatives (11a, 11c, 11e, 12b and 12c) were demonstrated to be more potent than known inhibitor MTH-Trp. Suzuki–Miyaura cross-coupling reaction of 11a–d with phenylboronic acid proceeded in high yields. In most cases, C5 and C6 substitutions on the corresponding indole ring were well tolerated. The tryptoline derivative 11c is a promising chemical lead for the discovery of novel IDO inhibitors.     

Nitro- and Amino-polysaccharide Formation from Dextran

Dextran was subjected to oxidative scission by periodate, followed by ring closure with nitromethane to form nitrodextran. The nitro group attached to the ring was reduced by LiAlH₄ to yield amino-polysaccharide of which the molecular weight was about 10,000. It became clear that nitrodextran consisted of 3-deoxy-3-nitro-mannopyranoside, -glucopyranoside, -galactopyranoside and -talopyranoside and their molar ratio was 6: 5: 1: 2 as determined by column Chromatographic separation and gas Chromatographic analysis of the methanolyzate of nitro-dextran.     

The daidzein- and estradiol- induced anorectic action in CCK or leptin receptor deficiency rats

We investigated the effect of daidzein feeding and estradiol treatment on food intake in cholecystokinin-1 receptor (CCK1R) deficiency, leptin receptor (ObRb) deficiency rats and their wild-type rats. These rats underwent an ovariectomy or a sham operation. For the 5 week experiment, each rat was divided in three groups: control, daidzein (150 mg/kg diet), and estradiol (4.2 μg/rat/day) groups. In both CCK1R+ and CCK1R? rats, daidzein feeding and estradiol treatment significantly decreased food intake. Daidzein feeding significantly reduced food intake in ovariectomized ObRb? rats, although not in ObRb+ rats. Estradiol treatment significantly lowered food intake in ovariectomized ObRb+ and ObRb? rats. In the ovariectomized rats, estradiol treatment significantly increases uterine weight, while daidzein feeding did not change it, suggesting that daidzein might have no or weak estrogenic effect in our experiment. These results suggest that CCK1R and ObRb signalings were not essential for the daidzein- and estradiol-induced anorectic action.     

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.     

Isolation and Constitution of a Xylan from Bamboo

A xylan from bamboo culm was isolated by extraction with aikali of chlorite holocellulose and fractional precipitation as a copper complex. The structure was investigated by means of examination of acid components by controlled hydrolysis, methylation analysis, and periodate oxidation. As a result, 4-O-methyl-α-D-glucuronic acid and 2-O-(4-O-methyl-α-D-glucopyranosyluronic acid) D-xylose were isolated and identified as acid components of the bamboo xylan. Hydrolysis of the fully methylated products afforded 2,3,5-tri-O- methyl-L-arabinose (1.6 moles), 2,3,4-tri-O-methyl-D-xylose (1.2 moles), 2,3,4,6-tetra-O-methyl-D-glucose(0.4 moles), 2,3-di-O-methyl-D-xylose (35.8 moles) and mono-O-methyl-D-xylose (2.6 moles). In addition to the above methylated sugars, 2,3,4-tri-O-methyl-D-glucuronic acid and partially methylated aldobiouronic acid were separated by cellulose column chromatography and identified. These results suggest that the bamboo xylan consists mainly of a linear backbone of 1,4-linked β-D-xylopyranose unit, to which L-arabinofuranose and 4-O-methyl-D-glucuronic acid were attached as a single side chain unit at C₂ or C₃.

Additional evidence for a linear chain structure has been given by periodate oxidation. On oxidation by periodate, the bamboo xylan consumed 1.09 moles of periodate and produced 0.05 mole of formic acid per anhydroxylose unit.     

Adaptation changes in dynamic postural control and contingent negative variation during repeated transient forward translation in the elderly

Background

Adaptation changes in postural control and contingent negative variation (CNV) for the elderly were investigated during repeated forward floor translation.

Methods

Fifteen healthy elderly persons, living in the suburban area of Kanazawa City, Japan, underwent backward postural disturbance by a forward-floor translation (S2) 2 s after an auditory warning signal (S1). A set with 20 trials was repeated until a negative peak of late CNV was recognized in the 600-ms period before S2, and the last set was defined as the final set. Electroencephalograms, center of foot pressure in the anteroposterior direction (CoPap), and electromyograms of postural muscles were analyzed.

Results

CoPap displacement generated by the floor translation was significantly decreased until the twelfth trial in the first set, and mean CoPap displacement was smaller in the second and final sets than in the first set. The mean displacement was significantly smaller in the final set than the previous set. A late CNV with a negative peak was not recognized in the first and second sets. However, most subjects (13/15) showed a negative peak by the fourth set, when the late CNV started to increase negatively from about 1,000 ms after S1 and peaked at about 300 ms before S2. At about 160 ms before the CNV peak, the CoPap forward shift started. The increase in timing of the gastrocnemius activity related to the CoPap shift was significantly correlated with the CNV peak timing (r = 0.64). After S2, peak amplitudes of the anterior postural muscles were significantly decreased in the final set compared to the first set.

Conclusions

It was demonstrated that even for the elderly, with so many repetitions of postural disturbance, a late CNV with a negative peak was recognized, leading to accurate postural preparation. This suggests the improvement of frontal lobe function (e.g., anticipatory attention and motor preparation) in the elderly.