Industrial production of (R)-1,3-butanediol by new biocatalysts

We have developed the economical and convenient biocatalytic process for the preparation of (R)-1,3-butanediol (BDO) by stereo-specific microbial oxido-reduction on an industrial scale. (R)-1,3-BDO is an important chiral synthon for the synthesis of various optically active compounds such as azetidinone derivatives lead to penem and carbapenem antibiotics.

We studied on two approaches to obtain (R)-1,3-BDO. The first approach was based on enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone; the second approach was based on enantio-selective oxidation of the undesired (S)-1,3-BDO in the racemate. As a result of screening for yeasts, fungi and bacteria, the enzymatic resolution of racemic 1,3-BDO by the Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-1,3-BDO with high enantiomeric excess and yield.

We characterized the (S)-1,3-BDO dehydrogenase purified from a cell-free extract of C. parapsilosis. This enzyme was found to be a novel secondary alcohol dehydrogenase (CpSADH). We have attempted to clone and characterize the gene encoding CpSADH and express it in Escherichia coli. The CpSADH activity of a recombinant E. coli strain was more than two times higher than that of C. parapsilosis. The production yield of (R)-1,3-BDO from the racemate increased by using the recombinant E. coli strain. Interestingly, we found that the recombinant E. coli strain catalyzed the reduction of ethyl 4-chloro-3-oxo-butanoate to ethyl (R)-4-chloro-3-hyroxy-butanoate with high enantiomeric excess.     

Metabolome analysis reveals the association between the kynurenine pathway and human herpesvirus 6 encephalopathy in immunocompetent children

Introduction

Human herpesvirus 6 (HHV-6) is the second most common causative pathogen of acute encephalopathy in immunocompetent children in Japan. Identification of biomarkers associated the pathophysiology is desirable to monitor disease severity, progression, and prognosis.

Objectives

To investigate potential biomarkers for HHV-6 encephalopathy, serum metabolome profiling was analyzed and candidate metabolites were investigated the function in the diseases.

Methods

Pediatric patients with HHV-6 encephalopathy (n?=?8), febrile seizure (n?=?20), and febrile infection without febrile seizure (n?=?7) were enrolled in this study, and serum metabolites were identified and quantified. For further analysis, serum samples of HHV-6 infected patients were analyzed by absolute quantification assay for kynurenine (KYN) and quinolinic acid (QUIN) in a total of 38 patients with or without encephalopathy. An in vitro blood–brain barrier (BBB) model was used to evaluate the effect of KYN and QUIN on BBB integrity because BBB damage induces brain edema.

Results

Metabolome profiling identified 159 metabolites in serum samples. The levels of KYN and QUIN, which belong to the tryptophan-KYN pathway, were significantly higher in the HHV-6 encephalopathy group than the other two groups. When quantified in the larger patient group, remarkably high levels of KYN and QUIN were observed exclusively in the encephalopathy group. Trans-endothelial electrical resistance of the BBB model was significantly decreased after QUIN treatment in culture.

Conclusion

Metabolome analysis revealed that KYN and QUIN may be associated with the pathophysiology of HHV-6 encephalopathy. In particular, QUIN may damage BBB integrity.

     

Ligand binding of the second PDZ domain regulates clustering of PSD-95 with the Kv1.4 potassium channel.

The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase ) domain. It binds to the N-methyl-D-aspartate glutamate receptor NR2 subunit or to the Shaker-type K(+) channel, Kv1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mDelta) and/or PDZ2 (PDZ2mDelta) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Kv1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mDelta) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (PSD-95:1mDelta-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mDelta was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Kv1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity.     

Epitope analysis of human monoclonal antibody specific for rice allergenic protein generated by in vitro immunization

We previously established an in vitro immunization protocol for generating antigen specific human monoclonal antibodies (mAbs). In vitro immunization was performed against the soluble protein of rice allergenic protein (RA), resulting in the generation of three B cell clones, AC7-1/F9, CB7-1/E2 and CB7-8/F5, all of which produce a RA-specific human monoclonal IgM antibody. We attempted to map the epitope regions recognized by thesem Abs to characterize their specificities. We performed two rounds of epitope mapping, rough mapping using 10-mer peptides covering the full-length RA with 5 amino acids overlapping, and fine mapping using 8-mer peptides covering the putative epitope regions from the rough mapping with 1amino acid overlapping. As a result of the fine mapping,we identified the epitope regions of these three mAbs as⁴⁵QVWQDCCRQ⁵⁴L, ⁵⁶AVDDGWCRCGA⁶⁷L and⁹¹FPGCRRG⁹⁸D on the RA molecule and found to be identical. Furthermore, we determined the putative core epitope regions, which are critical for mAb binding to each region, ⁴⁷WQDCC⁵²R and ⁶⁰GWC⁶³R. The information about the epitope region on the RA molecule,which might trigger the allergenic response, would be useful to establish a specific immunotherapy against rice allergy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improvement of a process for purification of tocopherols and sterols from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) is a useful material for purification of tocopherols and phytosterols (referred to as sterols). The SODD was first distilled, and the two substances were enriched. The preparation, which mainly contained free fatty acids (FFAs), sterols, and tocopherols, was named SODD tocopherols/sterols concentrate (SODDTSC). If sterols are converted to steryl esters and FFAs are converted to fatty acid methyl esters (FAMEs), relatively easy purification of tocopherols and steryl esters can be achieved because the boiling points of FAMEs, tocopherols, and steryl esters are different significantly. Hence, the development of a new two-step in situ reaction system was tried out for esterification of sterols with FFAs (first step) and esterification of FFAs with methanol (MeOH) (second step). A mixture of SODDTSC/water (95:5, w/w) and 250 units (U)/g-mixture of Candida rugosa lipase was prepared beforehand for the first-step reaction, and was agitated at 40 °C for 24 h with dehydration at 20 mmHg. Sterols were efficiently esterified, and the degree of esterification reached 95%. To the reaction mixture were added 7 M amounts of MeOH against unreacted FFAs, 20 wt.% water, and 25 U/g-mixture of Alcaligenes sp. lipase. The second-step reaction was then conducted at 30 °C for 20 h. Consequently, 95% FFAs were converted to FAME, and steryl esters synthesized by the first-step reaction were not reconverted to free sterols. Finally, SODDTSC (1.5 kg) was subjected to this two-step in situ reaction, and tocopherols and steryl esters were purified from the reaction mixture by short-path distillation. Tocopherols were purified to 72% (yield, 88%) and steryl esters were purified to 97% (yield, 97%).     

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.     

Use of Getah virus for antiviral assay of human interferon

Antiviral assay is used routinely for measuring the biological activity of interferon (IFN). However, the challenge viruses used in these assays are considered dangerous to the animal industry and pose a risk of human infection. For example, the vesicular stomatitis virus (VSV) is an important exotic disease agent in domestic animals, and the sindbis virus provokes rash, arthralgia, and fever in humans. Therefore, biosafety needs to be considered when antiviral assays are performed. We chose Getah virus as a candidate challenge virus because it is less hazardous to animals and humans. Crystal violet staining 50% CPE inhibition antiviral assay of human IFN using Getah virus was studied. Antiviral assay using Getah virus and FL cells gave a higher titer of human IFN than did assay using VSV. The titer of human IFN alpha was almost the same as that given by standardized control samples. The titer of human IFN by antiviral assay using Getah virus on the sheet method (IFN reacted the sheeted FL cells) was higher than those of the simultaneous reaction method (IFN reacted the suspending FL cells before sheeted). We therefore consider the sheet method useful for detection of small amounts of IFN. Antiviral assay using Getah virus on MDBK cells gave a lower titer of human IFN alpha than did assay using VSV. However, the adjusting the number of MDBK cells and the titer of Getah virus to get the best condition for CPE appearance, gave similar results in the assays using Getah virus and VSV. We consider that Getah virus is a potentially useful challenge virus for antiviral assay of human IFN.     

Sequence evolution of mitochondrial tRNA genes and deep-branch animal phylogenetics

Mitochondrial DNA sequences are often used to construct molecular phylogenetic trees among closely related animals. In order to examine the usefulness of mtDNA sequences for deep-branch phylogenetics, genes in previously reported mtDNA sequences were analyzed among several animals that diverged 20–600 million years ago. Unambiguous alignment was achieved for stem-forming regions of mitochondrial tRNA genes by virtue of their conservative secondary structures. Sequences derived from stem parts of the mitochondrial tRNA genes appeared to accumulate much variation linearly for a long period of time: nearly 100 Myr for transition differences and more than 350 Myr for transversion differences. This characteristic could be attributed, in part, to the structural variability of mitochondrial tRNAs, which have fewer restrictions on their tertiary structure than do nonmitochondrial tRNAs. The tRNA sequence data served to reconstruct a well-established phylogeny of the animals with 100% bootstrap probabilities by both maximum parsimony and neighbor joining methods. By contrast, mitochondrial protein genes coding for cytochrome b and cytochrome oxidase subunit I did not reconstruct the established phylogeny or did so only weakly, although a variety of fractions of the protein gene sequences were subjected to tree-building. This discouraging phylogenetic performance of mitochondrial protein genes, especially with respect to branches originating over 300 Myr ago, was not simply due to high randomness in the data. It may have been due to the relative susceptibility of the protein genes to natural selection as compared with the stem parts of mitochondrial tRNA genes. On the basis of these results, it is proposed that mitochondrial tRNA genes may be useful in resolving deep branches in animal phylogenies with divergences that occurred some hundreds of Myr ago. For this purpose, we designed a set of primers with which mtDNA fragments encompassing clustered tRNA genes were successfully amplified from various vertebrates by the polymerase chain reaction.Abbreviations AA stem amino acid-acceptor stem - AC stem anticodon stem - COI cytochrome oxidase subunit I - cytb cytochrome b - D stem dihydrouridine stem - MP maximum parsimony - mtDNA mitochondrial DNA - Myr million years - NJ neighbor joining - PCR polymerase chain reaction - Ti transition - T stem tC stem - Tv transversion Correspondence to: Y. Kumazawa     

Interleukin‐17A‐producing T lymphocytes in chronic active Epstein–Barr virus infection

T helper (Th) 17 cells are reportedly effector T cells that produce interleukin (IL)‐17A and play a significant role in the development of autoimmune diseases and immune responses for antimicrobial host defense. Production of IL‐17A in chronic active Epstein–Barr virus infection (CAEBV) was studied to investigate its contribution to pathogenesis of this disease. Significantly more IL‐17A‐producing cells were detected in the peripheral blood of CAEBV patients than in that of healthy controls, although a significant difference in serum IL‐17A production was not confirmed. Of the IL‐17A‐producing cells, 91.8% were cluster of differentiation (CD)4‐positive Th17 cells. Moreover, there were significantly more IL‐17A‐producing cells among CD4⁺ cells in peripheral blood of CAEBV patients than in that of controls (1.97 ± 0.69% vs. 1.09 ± 0.53%, P = 0.0073). These data suggest that IL‐17A‐producing cells may influence the pathophysiology of CAEBV.