Tetranor PGDM analyses for the amyotrophic lateral sclerosis: positive and simple diagnosis and evaluation of drug effect.

Amyotrophic lateral sclerosis (ALS) is a late-onset, progressive motor neuronal degenerative disease occurring as sporadically and as a familial disorder. The patients with ALS typically become progressively paralyzed and develop respiratory failure that eventually leads to death within 3-5years. For this disease, there is no effective diagnostic method and also drug. This report describes a simple and useful diagnostic biomarker for ALS. Our findings suggest that the combination analysis of a metabolite of prostaglandin D2, 11,15-dioxo-9-hydroxy-,2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM and tPGDM) with creatinine is the diagnostic approach for ALS with high accuracy. tPGDM has the potential to be an important diagnostic tool in the pre-symptomatic stages and progression evaluation of ALS, and also to be a biomarker for the evaluation of drug effect.

Participation in aflatoxin biosynthesis by a reductase enzyme encoded by vrdA gene outside the aflatoxin gene cluster.

Three reactions from hydroxyversicolorone to versicolorone, from versiconal hemiacetal acetate to versiconol acetate, and from versiconal to versiconol are involved in a metabolic grid in aflatoxin biosynthesis. This work demonstrated that the same reductase of Aspergillus parasiticus catalyzes the three reactions. The gene (named vrdA) encoding the reductase was cloned, and its sequence did not show homology to any regions in aflatoxin gene cluster. Its cDNA encoding a 38,566Da protein was separated by three introns in the genome. Deletion of the vrdA gene in A. parasiticus caused a significant decrease in enzyme activity, but did not affect aflatoxin productivity of the fungi. Although the vrdA gene was expressed in culture conditions conducive to aflatoxin production, it was expressed even in the aflR deletion mutant. These results suggest that the vrdA is not an aflatoxin biosynthesis gene, although it actually participates in aflatoxin biosynthesis in cells.

Phylogenetic construction of 17 bacterial phyla by new method and carefully selected orthologs.

Here, we constructed a phylogenetic tree of 17 bacterial phyla covering eubacteria and archaea by using a new method and 102 carefully selected orthologs from their genomes. One of the serious disturbing factors in phylogeny construction is the existence of out-paralogs that cannot easily be found out and discarded. In our method, out-paralogs are detected and removed by constructing a phylogenetic tree of the genes in question and examining the clustered genes in the tree. We also developed a method for comparing two tree topologies or shapes, ComTree. Applying ComTree to the constructed tree we computed the relative number of orthologs that support a node of the tree. This number is called the Positive Ortholog Ratio (POR), which is conceptually and methodologically different from the frequently used bootstrap value. Our study concretely shows drawbacks of the bootstrap test. Our result of bacterial phylogeny analysis is consistent with previous ones showing that hyperthermophilic bacteria such as Thermotogae and Aquificae diverged earlier than the others in the eubacterial phylogeny studied. It is noted that our results are consistent whether thermophilic archaea or mesophilic archaea is employed for determining the root of the tree. The earliest divergence of hyperthermophilic eubacteria is supported by genes involved in fundamental metabolic processes such as glycolysis, nucleotide and amino acid syntheses.

Comprehensive analysis of the origin of eukaryotic genomes.

There is currently no consensus on the evolutionary origin of eukaryotes. In the search of the ancestors of eukaryotes, we analyzed the phylogeny of 46 genomes, including those of 2 eukaryotes, 8 archaea, and 36 eubacteria. To avoid the effects of gene duplications, we used inparalog pairs of genes with orthologous relationships. First, we grouped these inparalogs into the functional categories of the nucleus, cytoplasm, and mitochondria. Next, we counted the sister groups of eukaryotes in prokaryotic phyla and plotted them on a standard phylogenetic tree. Finally, we used Pearson's chi-square test to estimate the origin of the genomes from specific prokaryotic ancestors. The results suggest the eukaryotic nuclear genome descends from an archaea that was neither euryarchaeota nor crenarchaeota and that the mitochondrial genome descends from alpha-proteobacteria. In contrast, genes related to the cytoplasm do not appear to originate from a specific group of prokaryotes.

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.

Truncated midkine correlates with sensitivity to anticancer drugs and malignancy in human tumor cell line.

Midkine (MK) is a heparin binding growth factor having functions of neurite-outgrowth, mitogenesis and tissue repair. This molecule is involved in tumor growth and metastasis. The MK molecule consists of five exons, but there is a truncated isoform, lacking exon 3. We established SW13 cells transfected with the human truncated MK cDNA. These cells were induced to undergo apoptosis by anticancer agents, cisplatin, etoposide (ETP), mitomycin C (MMC) and paclitaxel (PAX). Truncated midkine (tMK) suppressed cell death and helped the cells to be viable. When the cells were cultured on dishes coated with extracellular matrix molecules, spontaneous detachment occurred in the tMK expressing cells. Also tMK enhanced cell invasion. These results suggest that expression of tMK has cell-protective functions and plays important roles in carcinogenesis and malignancy. Furthermore, it is suggested that tMK has a greater ability of malignant transformation than full-length MK. Whether tMK is expressed or not will be useful information for improving cancer chemotherapy.

Truncated midkine induces transformation of cultured cells and short latency of tumorigenesis in nude mice.

To test whether truncated midkine (tMK), an alternative spliced form of exon 3, induces the transformation of mammalian cells, tMK cDNA was transfected into SW-13 cells. Although, the growth of MK transfectant (SW-13/MK) cells was close to those of the parent SW-13 and vector transfectant (SW-13/empty) cells, the growth of tMK transfectant (SW-13/tMK) cells was significantly promoted compared with that of the above three cells. Both SW-13/tMK and SW-13/MK formed colonies in 0.35% soft agar, indicating that tMK and MK induce mammalian cell transformation. SW-13/tMK frequently formed solid tumor after its subcutaneous injection into nude mice. Additionally, in SW-13/tMK and SW-13/MK-injected mice, advanced visible tumors were detected compared with that in the case of SW-13/empty-injected mice as control. These findings indicate that tMK induces mammalian cell transformation and promotes tumor establishment in vivo.

Alkali- and halo-tolerant catalase from Halomonas sp. SK1: overexpression in Escherichia coli, purification, characterization, and genetic modification.

A catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1, on the pKK223-3, was expressed in the catalase-lacking Escherichia coli strain UM2. Highly purified catalase showing a single band on SDS-PAGE was obtained by two liquid chromatography steps on DEAE-Toyopear1 and Chelating-Sepharose Fast Flow. The enzyme, oHktA, shows high catalase activity with a pH optimum at 10, and the activity was stable in 4 M KC1. This enzyme is thermo-sensitive, showing a significant loss of activity within 5 minutes at 37 degrees C. To modify the stability of the catalase, the addition of domain II of the heat stable Mn catalase from Thermus thermophilus to the C-terminus was made. When coexpressed with a chaperone (PhFKBP29) gene product, peptidyl-prolyl cis-trans isomerase, from a thermophilic bacterium, a chimeric catalase was produced in the soluble fraction. The stability of this catalase in the range of 37 degrees -45 degrees C was improved and it was stable for more than 1 h at 37 degrees C.

Exon skipping of midkine pre-mRNA is enhanced by intronic polymorphism in a colon cancer cell line.

A correlation between the polymorphism, heterogeneous G/T at the 62nd site of intron 3 in the midkine gene, and the induction of colorectal cancer has been reported [Cancer Lett. 180 (2002) 159]. The minigene containing exons 2, 3 and 4, as well as intronic sequences flanking exon 3, was transfected into COLO205 colon cancer cells. When the base of the site was G, correctly spliced mRNA was strongly detected. However in case of a G to T substitution, a truncated exon 3 mRNA was strongly detected. In this case, the detection of correctly spliced mRNA was weak. When the minigene was transfected into HCT-15 colon cancer cells, correctly spliced mRNA was strongly detected in the cases of both minigenes. This indicates the possibility that a G to T substitution at the 62nd site of intron 3 in the midkine gene enhances the expression of truncated midkine in colon cancer.

The origin of eukaryotes is suggested as the symbiosis of pyrococcus into gamma-proteobacteria by phylogenetic tree based on gene content.

Attempts were made to define the relationship among the three domains (eukaryotes, archaea, and eubacteria) using phylogenetic tree analyses of 16S rRNA sequences as well as of other protein sequences. Since the results are inconsistent, it is implied that the eukaryotic genome has a chimeric structure. In our previous studies, the origin of eukaryotes to be the symbiosis of archaea into eubacteria using the whole open reading frames (ORF) of many genomes was suggested. In these studies, the species participating in the symbiosis were not clarified, and the effect of gene duplication after speciation (in-paralog) was not addressed. To avoid the influence of the in-paralog, we developed a new method to calculate orthologous ORFs. Furthermore, we separated eukaryotic in-paralogs into three groups by sequence similarity to archaea, eubacteria (other than alpha-proteobacteria), and alpha-proteobacteria and treated them as individual organisms. The relationship between the three ORF groups and the functional classification was clarified by this analysis. The introduction of this new method into the phylogenetic tree analysis of 66 organisms (4 eukaryotes, 13 archaea, and 49 eubacteria) based on gene content suggests the symbiosis of pyrococcus into gamma-proteobacteria as the origin of eukaryotes.

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 and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus.

Nineteen fungi were tested for their ability to degrade aflatoxin B1 (AFB1). An extracellular enzyme from the edible mushroom Pleurotus ostreatus showed afaltoxin-degradation activity detected by thin-layer chromatography (TLC). An enzyme with this activity was purified by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. Optimum activities were found in the pH range between 4.0 and 5.0 and at 25 degrees C. Also, degradation activity of several dyes in the presence of H2O2 was tested, resulting in the detection of bromophenol blue-decolorizing activity. Based on these data, we suggest this enzyme is a novel enzyme with aflatoxin-degradation activity. Fluorescence measurements suggest that the enzyme cleaves the lactone ring of aflatoxin.

Presence of isochore structures in reptile genomes suggested by the relationship between GC contents of intron regions and those of coding regions.

Vertebrate genomes are mosaics of isochores. On the assumption that marked differences exist in the isochore structure between warm-blooded and cold-blooded animals, variations among vertebrates were previously attributed to adaptation to homeothermy. However, based on the data of coding regions from representatives of extant vertebrates, including a turtle, a crocodile (Archosauromorpha) and a few kinds of snakes (Lepidosauromorpha), it was recently hypothesized that the common ancestors of mammals, birds and extant reptiles already had the "warm-blooded" isochore structure. To test this hypothesis, the nucleotide sequences of alpha-globin genes including non-coding regions (introns) from two snakes, N. kaouthia and E. climacophora, were determined (accession number: AB104824, AB104825). The correlation between the GC contents in the introns and exons of alpha-globin genes from snakes and those from other vertebrates supports the above hypothesis. Similar analysis using data for exons and introns of other genes obtained from the GenBank (Release 131) also support the above hypothesis.

Characterization of subrepeat regions within rDNA intergenic spacers of the edible basidiomycete Lentinula edodes.

For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.

Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria supported by the newly clarified origin of functional genes.

In the previous report, we demonstrated the origin of eukaryotic cell nuclei as the symbiosis of Archaea in Bacteria by the newly developed "Homology-Hit Analysis". In that case, we counted yeast Open Reading Frames (ORFs) showing the highest similarity to a bacterial ORF as orthologous ORFs (Orthologous ORFs were produced by speciation from a common ancestor, and have the highest similarity to each other.) by comparing whole ORFs of yeast with those of individual bacteria. However, we could not count all yeast ORFs showing the highest similarity to a bacterial ORF in functional categories of yeast. Therefore, the origin of ORFs in the functional categories of yeast could not be inferred strictly. Here, we have improved the method for detecting orthologous ORFs. In this method, we count the numbers of ORF with the highest similarity between individual yeast functional categories and individual bacteria as orthologous ORFs. By this method, it was possible to detect the correct orthologous ORFs and to infer the origins of the functional categories in eukaryotic cells. As a result, two categories, assembly of protein complexes and DNA repair were newly judged to be of Archaeal origin, while five categories, lipid (fatty-acid and isoprenoid) metabolism, protein folding and stabilization, signal transduction, organization of the plasma membrane and organization of the cytoplasm, were newly judged to be of Bacterial origin. On the other hand, the origins of two categories (meiosis and cellular import, which were determined in the previous analysis) could not be judged. It is considered that functional categories related to the nucleus have origins common to Archaea, while those related to the cytoplasm have origins common to Bacteria. From these data including the origin of plasma membrane, it was further clarified that cell nucleus originated by the symbiosis of Archaea in Bacteria.

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.

Association of an intronic polymorphism in the midkine (MK) gene with human sporadic colorectal cancer.

Midkine (MK) is a heparin-binding growth factor specified by a retinoic acid responsive gene. It plays important roles in development and carcinogenesis. The MK gene is located on chromosome 11q11.2 in humans. A heterozygous G to T transition at the 62nd base in intron 3 of this gene has been identified in sporadic colorectal and gastric cancers (Int. J. Mol. Med. 6 (2000) 281). To clarify whether this polymorphism is associated with a cancer risk, a case-control study was conducted. We examined 98 colorectal, 60 gastric, 59 esophagus, 32 lung and 37 breast cancer tissue specimens and their corresponding non-neoplastic tissues. Also, 86 unaffected control specimens were examined. The G/T genotype frequency in colorectal cancers was higher than that in normal samples (11.2 versus 2.3%; P=0.017). Therefore, this genotype could represent a risk factor for tumorigenesis in the colon and rectum of Japanese.

Purification, characterization, and gene sequencing of a catalase from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1.

An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4% yield of the catalase was obtained with 97% purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH2OH, NaN3, and 3-amino-1,2,4-triazole, active at pH 5.0-11.0, thermo-sensitive, and KCl-tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99% identity with that of Vibrio rumoiensis S-1T.

Monoclonal antibody to stage-specific fetal brain 68-kDa glycoprotein (FGP68) revealed increased FGP68 expression in human primary brain tumors.

We have produced a novel rat IgG(2a) monoclonal antibody against a stage-specific fetal brain glycoprotein of 68 kDa (FGP68), and succeeded in applying it to staining paraffin sections. To gain some insight into the pathobiological significance of this FGP68, this monoclonal antibody was used in immunohistochemical studies to compare the expression of FGP68 and Ki-67 antigen (MIB-1) in 235 primary brain tumors. Approximately half of the glioblastomas multiforme (GBMs) (44/75) and anaplastic astrocytomas (9/17) as well as some astrocytomas (5/30), medulloblastomas (2/14) and primitive neuroectodermal tumors (2/10) had tumor cells that expressed FGP68; however, pilocytic astrocytomas (0/7), oligodendrogliomas (0/15), ependymomas (0/6), schwannomas (0/21), meningiomas (0/22) and pituitary adenomas (0/18) did not express FGP68. The values of the MIB-1 labeling index were statistically higher in GBMs (0.005< P<0.01, Wilcoxon rank-sum test) and anaplastic astrocytomas (0.025< P<0.05) that expressed FGP68 than in those that did not. Normal brain tissue from 20 individuals aged 3-75 years was negative for FGP68 and MIB-1. We conclude that primary brain tumors express FGP68, one of the oncofetal proteins derived from fetal brain, and that FGP68 expression in certain brain tumor cells may depend, in part, on proliferation potential. Based on the possibility that the stage-specific FGP68 plays an important role in brain embryogenesis, some of FGP68-expressing tumor cells might phylogenetically revert to more primitive cells.

Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis

The presence of rhodopsin-like proteins in the eyes and auricles of the freshwater planarian Dugesia japonica was confirmed using anti-frog-rhodopsin rabbit IgG. The apparent relative molecular masses of these proteins were 65x10(3) and 62x10(3), and positive reactions to IgG were localized to the microvilli of the photoreceptor cells in the eyes and to the sensory cilia, rootlets and microvilli in the auricles. Eye- or head-excised planarians showed no negative phototaxis, whereas intact or auricle-excised planarians did. During regeneration in head-excised planarians, the appearance of rhodopsin-like proteins in the regenerating eyes corresponded to the recovery of negative phototaxis behaviour. Head or auricle excision enhanced asexual fission under continuous illumination. However, eye excision had no such effect. These results suggest that the rhodopsin-like proteins in the eyes work as photoreceptors for negative phototaxis behaviour and that, in the auricles, they are involved in asexual fission originating from the circadian rhythm.