Repellents in the Japanese cedar,Cryptomeria japonica,against the pill-bug,Armadillidium vulgare

Sandaracopimarinol and (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one were isolated and identified from Cryptomeria japonica as repellents against Armadillidium vulgare which is well known as an unpleasant pest in the house and as vegetable pest in Japan. These compounds strongly repelled A. vulgare when they were combined, although each compound alone did not show any activity.     

Accurate measurement of near-micromolar oxygen concentrations in aqueous solutions based on enzymatic extradiol cleavage of 4-chlorocatechol: applications to improved low-oxygen experimental systems and quantitative assessment of back diffusion of oxygen from the atmosphere

An enzymatic method for measuring the O(2) concentrations of aqueous solutions was developed by involving 4-chlorocatechol and catechol 2,3-dioxygenase from Pseudomonas putida. With this system, the amount of O(2) in a sample solution can be measured as the amount of 5-chloro-2-hydroxymuconate semialdehyde formed through the enzyme reaction. The product was stable and its anion exhibited strong absorption around 380 nm (molar absorption coefficient of 4.3 x 10(4) M(-1) cm(-1), pK value of 5.4). A sensitive HPLC method involving a BioAssist Q column was developed to individually quantify the products derived from 4-chlorocatechol and catechol. When the O(2) concentration in a sample solution sealed in a vial was lowered from the air-saturation level by means of the amount enzymatically reacted with a known amount of catechol, the concentration of remaining O(2) could be successfully measured by the HPLC method. We developed devices through which reagents could be added to solutions sealed in cuvettes or the vessel of an oxygen electrode system under a flow of argon. By applying these devices, the submicromolar O(2) concentration of an anoxic solution and the back diffusion of O(2) from the atmosphere could be directly determined for the first time. The K(m) values of the dioxygenase and an ascorbate oxidase for oxygen were also determined to be 7.2 (at pH 7.5) and 114 microM (at pH 6.5), respectively, at 25 degrees C.     

Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes

Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.     

Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity

Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.     

Extensive analysis of ORF sequences from two different cichlid species in Lake Victoria provides molecular evidence for a recent radiation event of the Victoria species flock: identity of EST sequences between Haplochromis chilotes and Haplochromis sp. "Redtailsheller"

The Lake Victoria Cichlid fishes have diverged very rapidly. The estimated 500 species inhabiting the lake are believed to have arisen within the last 14,000 years. The fishes' jaws and teeth have diverged markedly to adapt to different feeding behaviors and environments. To examine how the genomes of these fishes differentiated during speciation, we performed comparative analysis of expressed sequenced tag (EST) sequences. We constructed cDNA libraries derived only from the jaw portions of two cichlid species endemic to Lake Victoria. We sequenced 17,280 cDNA clones from Haplochromis chilotes and 9600 cDNA clones from Haplochromis sp. "Redtailsheller" and obtained 543 different genes common to both species. Of these genes, 441 were essentially identical between species and 102 contained base replacements in their open reading frame (ORF) or untranslated (UTR) regions. Comparative analysis of 71 selected sequences has revealed that while the degree of polymorphism is 0.0054/site for H. chilotes and 0.0047/site for H. sp. "Redtailsheller", genetic distance between the two species is 0.0031/site. The genetic distance particularly indicates that the two species diverged about 890,000 years ago.     

Molecular chaperones: proposal of a systematic computer-oriented nomenclature and construction of a centralized database

Molecular chaperones are a wide group of unrelated protein families whose role is to assist others proteins. Comparably, under environmental stress, stress proteins behave as biocatalysts of protein stabilization. Stress proteins include a large class of proteins that were originally termed heat shock proteins (HSPs) due to their initial discovery in tissues exposed to elevated temperatures. Many, but not all, stress proteins and HSPs are molecular chaperones. Moreover, not all HSPs are derivable from stress. HSPs are structurally diversified by the contribution of various domains having specific roles. HSPs have been grouped, mainly on the basis of their molecular masses, into specific families that include small HSPs (sHSPs)/alpha-crystallins, HSP10s, HSP40s, HSP60s, HSP70s, HSP90s, HSP100s and HSP110s. The names of these major families are historical artefacts with limited information content. Using the current databases, names and proteic domains of many molecular chaperones in different species were analyzed. Although traditional names of HSPs are trivial, it is unrealistic to suggest replacing them, because they are preferred and widely used. Here we suggest that these traditional names be chaperoned, in silico, by a systematic nomenclature. Thus, for example, with the same intent of use of [trioxygen: O3] for ozone, we propose here C7HSP70[Ehsa]ER-P11021 for GRP78 (78 kDa endoplasmic Human molecular chaperone in HSP70 superfamily with P11021 as its accession number in the database of the National Center for Biotechnology Information (NCBI)). The proposed systematic computer-oriented naming and classification method is designed for HSPs and also their partners based on the number of amino acids, domain structure, phylogenetic domain, localization in the cell and accession number as stated in the NCBI. Arabidopsis thaliana was analyzed as a model, because it contains a large number of various HSPs localized in several organelles. Overall, this naming system helps in building, optimizing and managing a novel online database entirely devoted to HSPs. The purported taxonomy, coupled with the newly constructed database, can contribute to studies involving large amounts of stored data on HSPs.     

Purification of a low-molecular-weight phospholipase A(2) associated with soluble high-molecular-weight acidic proteins from rabbit nucleus pulposus and its comparison with a rabbit splenic group IIa phospholipase A(2)

An intervertebral disc is a large peice of avascular cartilage rich in proteoglycans and water consisting of gelatinous nucleus pulposus and fibrous annulus fibrosus. The soluble fraction of rabbit nucleus pulposus exhibited unusually high Ca(2+)-dependent phospholipase A(2) (PLA(2)) activity (about 70% of the total PLA(2) activity). The soluble PLA(2) activity was 6-7-fold higher than those of rabbit annulus fibrosus and spleen. The PLA(2) was bound to an anion-exchange column at pH 7.4, and eluted near the void volume as a broad peak on gel-filtration on a TSKgel SuperSW3000 column developed with a buffer containing 0.1-0.2 M salt. When the gel-filtration column was developed in the presence of 1 M salt, almost all the PLA(2) activity was eluted near the total available volume. The soluble PLA(2) was purified to near homogeneity. A Ca(2+)-dependent PLA(2) was also purified from the fractions extracted with 1 M KBr from nucleus pulposus. For comparison, we purified a Ca(2+)-dependent PLA(2) from the KBr fraction of spleen. The splenic PLA(2) was identical to a group IIa PLA(2), as judged from its N-terminal amino acid sequences and mass spectra. On SDS-polyacrylamide gel electrophoresis the enzymes purified from the soluble and KBr fractions of nucleus pulposus both gave a major 15. 7-kDa band at the same position as splenic group IIa PLA(2). These results suggest that group IIa PLA(2) is associated with soluble high-molecular-weight proteins, most likely proteoglycans, in the extracellular matrix of rabbit nucleus pulposus.     

Isolation and identification of the probing stimulants in the rice plant for the white-back planthopper, Sogatella furcifera (homoptera: delphacidae)

Adult females of the white-back planthopper, Sogatella furcifera, showed characteristic behavior of stylet sheath deposit on a parafilm membrane when fed on a 2% aqueous crude rice leaf and stem extract containing 15% sucrose. Subsequent bioassays revealed that the butanol-soluble fraction of the extract was highly active against the insects. When the butanol fraction was chromatographed on an ODS open column and eluted in sequence with a mixture of an increasing concentration of methanol in water, the 40 % methanol fraction was separated as the most active. A further bioassay of the HPLC components in the active fraction revealed that two major components (1 and 3) stimulated the high probing activity of the white-back planthopper only when they were combined. Of the active components, one component (3) was identified to be tricin 5-O-glucoside by spectroscopic analyses.     

Determination of whole prokaryotic phylogeny by the development of a random extraction method

The construction of accurate prokaryotic phylogeny is important not only in the field of evolutionary biology, but also in microbiology and pathology. However, in constructing a phylogenetic tree to trace prokaryotic evolution, the phylogenetic relationship is often changed by the choice of species. For the estimation of the accurate lineage of prokaryotes, a new method, named the "random extraction method", was developed. In this method, 16S rRNA sequence data were randomly extracted 1000 times from each closely-related taxa such as seven phyla of Eubacteria and one domain of Archaea and phylogenetic trees were constructed by the data to clarify the relationship of those groups. Next, the tree topology was counted and the most supported tree topology was found as the most plausible phylogenetic tree. To evaluate the reliability of each node, we developed the "Branching rate" (BR) and calculated for every tree. And also, computational simulation analysis was carried out to confirm these methods. On the assumption that the root of life is between Archaea and Eubacteria, the obtained phylogenetic relationships of phyla are the following. At first, Archaea (Euryarchaeota, Crenarchaeota and Korarchaeota) diverged, and Thermotogales, Cyanobacteria and Chlamydiales diverged in this order, then Firmicutes (Actinobacteria and Bacillus/Clostridium group cluster) and Proteobacteria (alpha and beta/gamma cluster) diverged. In addition, it was shown by the BR that the position of the node of Firmicutes Actinobacteria and Firmicutes Bacillus/Clostridium was changeable for each extraction. Therefore, it was suggested that the differences among the phylogenetic trees of prokaryotes were caused by the influence of these phyla.     

Comparative phylogeography of three Leptocarabus ground beetle species in South Korea, based on the mitochondrial COI and nuclear 28S rRNA genes

We analyzed the intraspecific gene genealogies of three Leptocarabus ground beetle species (L. seishinensis, L. semiopacus, L. koreanus) in South Korea using sequence data from the mitochondrial cytochrome oxidase subunit I (COI) and nuclear 28S rRNA (28S) genes, and compared phylogeographical patterns among the species. The COI data detected significant genetic differentiation among local populations of all three species, whereas the 28S data showed genetic differentiation only for L. seishinensis. The clearest differentiation of L. seishinensis among local populations was between the northern and southern regions in the COI clades, whereas the 28S clade, which likely indicates relatively ancient events, revealed a range expansion across the northern and southern regions. Leptocarabus semiopacus had the most shallow differentiation of the COI haplotypes, and some clades occurred across the northern and southern regions. In L. koreanus, four diverged COI clades occurred in different regions, with partial overlaps. We discuss the difference in phylogeographical patterns among these Leptocarabus species, as well as between these and other groups of carabid beetles in South Korea.