Chemical Modification of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Tyrosyl Residues Iodinated

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153—159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.     

Genetic Variation and Population Differentiation in Natural Populations of Cryptomeria japonica

Abstract Sugi (Cryptomeria japonica D. Don) is a valuable tree species in Japan. The present natural distribution is limited to small scattered areas in temperate moist regions, and most of these areas are surrounded by vast artificial plantations. We studied natural populations of C. japonica in an effort to determine the amount and distribution of genetic diversity using 12 allozyme markers. The amount of genetic variation within the species is high (H_T=0.196) but most is found within populations with little among populations (G_ST=0.034) despite their isolated distribution. This pattern of genetic diversity is inferred to be the consequence of the following: (1) the distribution of this species in the past was wider and more continuous than it is now; (2) a high rate of gene flow occurs, perhaps including gene flow between natural populations and plantations; and (3) the long lifespan. However, the distribution of allele frequencies at the 6Pg-1 in northern populations on the side near the Sea of Japan is clearly different from those in other populations. This observation is inferred to result from founding events.     

Changes in magnetic resonance images in human skeletal muscle after eccentric exercise

To investigate the time-course of changes in transverse relaxation time (T₂) and cross-sectional area (CSA) of the quadriceps muscle after a single session of eccentric exercise, magnetic resonance imaging was performed on six healthy male volunteers before and at 0, 7, 15, 20, 30 and 60 min and 12, 24, 36, 48, 72 and 168 h after exercise. Although there was almost no muscle soreness immediately after exercise, it started to increase 1 day after, peaking 1–2 days after the exercise (P<0.01). Immediately after exercise, T₂ increased significantly in the rectus femoris, vastus lateralis and intermedius muscles (P<0.05) and decreased quickly continuing until 60 min after exercise. At and after the 12th h, a significant increase was perceived again in the T₂ values of the vastus lateralis and intermedius muscles (P<0.01) [maximum 9.3 (SEM 2.8)% and 10.9 (SEM 2.2)%, respectively]. The maximal values were exhibited at 24–36 h after exercise. In contrast, the rectus femoris muscle showed no delayed-stage increase. Also, in CSA, an increase after 12 h was observed in addition to the one immediately after exercise in the vastus lateralis, intermedius and medialis and quadriceps muscles as a whole (P < 0.01), reaching the maximal values at 12–24 h after exercise. The plasma creative kinase activity remained unchanged up to 24 h after and then increased significantly 48 h after exercise (P < 0.05). Beginning 12 h after exercise, the subjects whose T₂ and CSA increased less than the others displayed a faster decrease in muscle soreness. These results suggested that T₂ and CSA displayed bimodal responses after eccentric exercise and the time-courses of changes in them were similar to those in muscle soreness.     

Discovery of a pimaricin analog JBIR-13, from <Emphasis Type="Italic">Streptomyces bicolor</Emphasis> NBRC 12746 as predicted by sequence analysis of type I polyketide synthase gene

Sequence analysis of ketosynthase domain amplicons from Streptomyces bicolor NBRC 12746^T revealed the presence of previously unreported type I polyketide synthases (PKS-I) genes. The clustering of these genes with the reference PKS-1 sequences suggested the possibility to produce a polyene compound similar to pimaricin. Thus, the cultured sample from NBRC 12746^T was analyzed for the production of polyene compounds. The strain produced an antifungal compound which displayed the UV absorption spectrum of tetraene macrolides. The structure determination based on the spectroscopic analysis of the purified compound resulted in the identification of a novel pimaricin analog JBIR-13 (1). This study therefore strongly suggested that a careful analysis of PKS-I genes can provide valuable information in the search of novel bioactive compounds within a class predicted from phylogenetic analysis. H. Komaki and M. Izumikawa contributed equally to this work.     

Gene Expression Pattern after Insertion of Dexamethasone-Eluting Electrode into the Guinea Pig Cochlea

A cochlear implant is an indispensable apparatus for a profound hearing loss patient. But insertion of the electrode entails a great deal of stress to the cochlea, and may cause irreversible damage to hair cells and related nerve structure. Although damage prevention effects of dexamethasone have been reported, long-term administration is difficult. In this study, we used a dexamethasone-eluting electrode in the guinea pig cochlea, and compared the gene expression after 7 days insertion with that of a normal electrode and non-surgically treated control by microarray. 40 genes were up-regulated 2-fold or more in the normal electrode group compared to the non-surgically treated group. Most of the up-regulated genes were associated with immune response and inflammation. In the dexamethasone-eluting group, compared to the normal electrode group, 7 of the 40 genes were further up-regulated, while 12 of them were down-regulated and there was a tendency to return to the non-surgical condition. 9 genes were down-regulated 2-fold or less with normal electrode insertion, and 4 of the 9 tended to return to the non-surgical condition in the dexamethasone-eluting group. These genes are certainly involved in the maintenance of the physiological functions of the cochlea. Our results indicate that the dexamethasone-eluting electrode will have an effect on the normalization of homeostasis in the cochlea.     

Global searches for microalgae and aquatic plants that can eliminate radioactive cesium, iodine and strontium from the radio-polluted aquatic environment: a bioremediation strategy

The Fukushima 1 Nuclear Power Plant accident in March 2011 released an enormously high level of radionuclides into the environment, a total estimation of 6.3 × 10¹⁷ Bq represented by mainly radioactive Cs, Sr, and I. Because these radionuclides are biophilic, an urgent risk has arisen due to biological intake and subsequent food web contamination in the ecosystem. Thus, urgent elimination of radionuclides from the environment is necessary to prevent substantial radiopollution of organisms. In this study, we selected microalgae and aquatic plants that can efficiently eliminate these radionuclides from the environment. The ability of aquatic plants and algae was assessed by determining the elimination rate of radioactive Cs, Sr and I from culture medium and the accumulation capacity of radionuclides into single cells or whole bodies. Among 188 strains examined from microalgae, aquatic plants and unidentified algal species, we identified six, three and eight strains that can accumulate high levels of radioactive Cs, Sr and I from the medium, respectively. Notably, a novel eustigmatophycean unicellular algal strain, nak 9, showed the highest ability to eliminate radioactive Cs from the medium by cellular accumulation. Our results provide an important strategy for decreasing radiopollution in Fukushima area.     

Enzymatic measurement of phosphatidic acid in cultured cells

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 µM, and the detection limit was 5 µM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.     

Essential Role of Neuron-Enriched Diacylglycerol Kinase (DGK), DGKβ in Neurite Spine Formation,Contributing to Cognitive Function

Background

Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKβ is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated.

Methodology/Principal Findings

We, therefore, developed DGKβ KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKβ. In addition, overexpression of DGKβ in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKβ, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKβ but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed.

Conclusions/Significance

These results demonstrate that membrane-localized DGKβ regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory.