Conformations of helical Aib peptides containing a pair of l‐amino acid and d‐amino acid

A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, ¹H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 3₁₀‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 3₁₀‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

Epistasis effects of COMT and MTHFR on inter-individual differences in mental health: Under the inverted U-shaped prefrontal dopamine model

Higher cognitive performance, maintenance of mental health and psychological well-being require adequate prefrontal cortex (PFC) function. “Inverted U-shaped” dopamine model indicates optimal PFC dopamine level is important to attain its function while high or low levels have adverse effects. Catechol-O-methyltransferase (COMT) and methylenetetrahydrofolate reductase (MTHFR) may be involved in this complex non-linear PFC dopamine regulation. We addressed whether genetic variation reflecting COMT and MTHFR activities can explain the inter-individual mental health differences in healthy Japanese men (n = 188). The mental health was measured by Mental Health Inventory (MHI)-5 score. The rs4633–rs4818–rs4680 haplotypes were used to represent the multilevel COMT activities, while for MTHFR, the functional single polymorphism, rs1801133 (C677T), was used. We examined the effectiveness of haplotype-based association analysis of COMT on mental health together with studying its interaction with MTHFR-C677T. As a result, the relation between activity-ranked COMT genotype and MHI-5 score showed a tendency to fit into an “inverted U-shaped” quadratic curve (P = 0.054). This curvilinear correlation was significant in the subjects with MTHFR-CC (P < 0.001), but not with MTHFR T-allele carriers (P = 0.793). Our pilot study implies a potential influence of COMT and MTHFR genotypic combination on normal variation of mental health.     

Automated system for high-throughput protein production using the dialysis cell-free method

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).     

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.     

Prevalence of periodontopathic bacteria on the tongue dorsum of elderly people

Objectives: The aim of this study was to analyse the prevalence of oral bacteria on the dorsum of the tongue. In addition, the relationship between the number of teeth and the microflora present on the coating of the tongue in a population of 85‐year‐old people was assessed. Subjects and methods: Two hundred and five individuals (89 males, 116 females) from the same geographical area who were 85 years of age were examined. Five periodontopathic bacteria (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Treponema denticola) and one cariogenic bacterium (Streptococcus mutans) were analysed using a polymerase chain reaction assay of tongue samples from the population. Results: Periodontal bacteria‐positive individuals have more teeth than that of periodontal bacteria‐negative people. Between the periodontal bacteria‐positive and ‐negative individuals, there were significant differences in the mean number of teeth for P. gingivalis (p < 0.0001), T. denticola (p < 0.001), F. nucleatum (p = 0.002), and T. forsythia (p = 0.005), while there were no significant differences for A. actinomycetemcomitans (p = 0.998) or S. mutans (p = 0.147). Conclusions: A wide range of species, including anaerobes, was detected in 85‐year‐old subjects. It was found that the detection of periodontal bacteria on the tongue coating increased with the number of teeth. There was a positive relationship between the tooth number and periodontopathic bacteria, except for A. actinomycetemcomitans.These results suggest that tongue care is essential for preventing oral disease and needs to be part of any oral care programme in elderly people.     

Autophagy and neurodegeneration

The proteasome and lysosome are sophisticated apparatuses capable of shredding unnecessary proteins in eukaryotic cells. The proteasome and its partner ubiquitin (which functions as a destination signal for proteolysis) play crucial roles in selective breakdown of not only short-lived regulatory proteins but also abnormal proteins that need to be rapidly eliminated from the cells. It is generally accepted that deficits of the proteasome-ubiquitin system are associated with various neurodegenerative diseases, since ubiquitin-positive inclusions frequently appear in neurons of patients and mice models of neurodegenerative diseases. However, investigators working in the field of neuronal diseases have focused their attention in recent years on autophagy (Greek for "the eating of oneself") following the recent discovery that ablation of autophagy leads to accumulation of ubiquitin-positive inclusions, which are the pathological hallmark of neurodegenerative diseases. Here we discuss the consequences of autophagy deficiency in neurons.     

Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides

The present study includes the exploration of new possible nucleoside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides (4a–g), which were synthesized by 10–15 synthetic steps and characterized adequately. We report the anti-HCV activities and cytotoxicities of 4a–g. Compound 4a was analyzed by single crystal X-ray diffraction which showed some puckering in the cyclopentene ring with a 2′-endo conformation and anti-base disposition (χ = ?125.7°).     

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation

Water‐insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α‐1,3‐glucanase) and dextranase (α‐1,6‐glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi‐functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE‐SUMOstar Amp plasmid vector. The resultant his‐tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non‐adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol‐sulfuric acid method, and reducing sugars were quantified by the Somogyi–Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose‐dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.