One-pot chemo-enzymatic enantiomerization of racemates

A new one-pot chemo-enzymatic procedure was developed for enantiomerization of racemates based on enzymatic enantiospecific oxidation of a substrate and chemical non-enantiospecific reduction of the product. The principle is shown as follows for the -proline production.

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-Proline and -pipecolate were produced from racemic proline and pipecolate by means of amino acid oxidase and sodium borohydride in high yield in this reaction system [J.W. Huh, K. Yokoigawa, N. Esaki, K. Soda, Biosci., Biotechnol., Biochem. 56 (1992) 2081]. - and -Lactate were -enantiomerized in a one-pot reaction system containing -lactate oxidase and sodium borohydride in the similar manner [S. Mukoyama, K. Yamanaka, T. Oikawa, K. Soda, Nippon Nogei Kagaku Kaishi 73 (1999) 62]. Pyruvate was also converted to an equimolar amount of lactate in the same system. α-Hydroxybutyrate can be produced from the - and -isomers, and α-ketobutyrate in the same manner though slowly. This method is applicable to production of other chiral compounds from the corresponding racemates.     

Congenital synostosis of the fourth and fifth metacarpals treated by free bone grafting from the fusion site

Simultaneous correction of the alignment and the shortening of the deformed little finger in congenital synostosis of the fourth and fifth metacarpals was accomplished by insertion of a wedge-shaped bone block harvested from the fusion site following transverse osteotomy of the fifth metacarpal. Correction can be performed simply and successfully without morbidity of the donor site when this technique is applied to suitable patients.     

Cyclin E as a coactivator of the androgen receptor

Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy.     

Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity

Metastin has been identified as a metastasis suppressor gene product that mediates its function through a G protein coupled receptor, GPR54. To refine insight into the critical pharmacophore for the activation of GPR54, we have conducted alanine and d-amino acid scanning on a biologically active metastin fragment (45-54). Based on these data and structures of peptides previously reported to activate GPR54, a series of shortened metastin (45-54) derivatives were synthesized and tested for the ability to induce GPR54 signaling. These biological experiments were performed in yeast containing human GPR54 that was coupled to the pheromone response pathway and a pheromone responsive lacZ reporter gene. Compounds 32, 33, and 39, which possess an N-terminal basic group and a C-terminal RW-amide motif, were strong agonists, similar to the level of metastin. This may provide an approach to reverse the pro-metastatic effect of metastin deletion in multiple malignant tumors.     

An isocratic HPLC method for the simultaneous determination of novel stable lipophilic ascorbic acid derivatives and their metabolites

2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

Sustained brain-derived neurotrophic factor up-regulation and sensorimotor gating abnormality induced by postnatal exposure to phencyclidine: comparison with adult treatment

Brain-derived neurotrophic factor (BDNF) is involved in synaptic development and plasticity, and alterations in BDNF expression or signaling are implicated in drug addiction and psychiatric diseases, such as depression and schizophrenia. In this study, we administered phencyclidine to postnatal and adult rats with different time schedules, and determined the correlations between BDNF expression and the behavioral effects. Both single and repeated phencyclidine injections into adult rats induced BDNF up-regulation in the corticolimbic system and a decrease in prepulse inhibition, both of which were transient. In contrast, subchronic postnatal administration increased BDNF protein and mRNA levels in the hippocampus and entorhinal cortex, which were sustained until 8 weeks of age. In parallel, the postnatal rats treated with phencyclidine developed a persistent decrease in prepulse inhibition at the adult stage. The chronic BDNF increase appeared to contribute to the prepulse inhibition abnormality, as subchronic BDNF infusion into the hippocampus of normal rats mimicked the prepulse inhibition deficits. This study suggests that phencyclidine exposure during brain development induces sustained BDNF up-regulation in the limbic system with a biological link to sensorimotor gating deficits.     

Role of gut-associated lymphoreticular tissues in antigen-specific intestinal IgA immunity

This study assessed the roles of the postnatal lymphotoxin-beta receptor (LTbetaR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTbetaR-dependent events by postnatal administration of the fusion protein of LTbetaR and IgG Fc (LTbetaR-Ig) reduced both the size and numbers of Peyer's patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTbetaR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTbetaR-Ig eliminated the formation of ILF. LTbetaR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTbetaR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.     

Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in-vitro de-epoxidation in monogalactosyldiacylglycerol micelles

The dynamics of the xanthophyll cycle relative to non-photochemical quenching (NPQ) were examined in tobacco plants overexpressing violaxanthin de-epoxidase (VDE), PsbS and PsbS+VDE for effects on NPQ and violaxanthin (V) de-epoxidation over a range of light intensities. Induction of de-epoxidation and NPQ increased in overexpressed VDE and PsbS plants, respectively. Surprisingly, under low light, overexpressing PsbS enhanced de-epoxidation in addition to NPQ. The effect was hypothesized as due to PsbS binding zeaxanthin (Z) or inducing the binding of Z within the quenching complex, thus shifting the equilibrium toward higher de-epoxidation states. Studies in model systems show that Z can stereospecifically inhibit VDE activity against violaxanthin. This effect, observed under conditions of limiting lipid concentration, was interpreted as product feedback inhibition. These results support the hypothesis that the capacity of the thylakoid lipid phase for xanthophylls is limited and modulates xanthophyll-cycle activity, in conjunction with the release of V and binding of Z by pigment-binding proteins. These modulating factors are incorporated into a lipid-matrix model that has elements of a signal transduction system wherein the light-generated protons are the signal, VDE the signal receptor, Z the secondary messenger, the lipid phase the transduction network, and Z-binding proteins the targets.     

Construction and characterization of chimeric proteins composed of type-1 and type-2 periplasmic binding proteins MglB and ArgT

The respective type-1 and type-2 periplasmic binding proteins (PBPs) MglB and ArgT are believed to have evolved from a common ancestor into siblings showing topological differences in their main chain connectivity. At first glance, they show similar structure. But, more detailed examination reveals that the chain connectivity of ArgT is more convoluted than that of MglB. Reflecting that complexity, the folding of ArgT is complicated and involves intermediate folds. On the other hand, the folding of MglB is a simple two-state transition. In the present study, we constructed and characterized several chimeras made up of various subdomains of MglB and ArgT with the aim of gaining insight into the evolution of protein folding and protein structure. Although these chimeras did not fold as compactly as their parental proteins, some did exhibit cooperative folding, which suggests that novel proteins with new connectivity and new folding pathways could have emerged at a fairly high rate throughout the evolution of proteins.