Molecular phylogeny of osteoglossoids: a new model for Gondwanian origin and plate tectonic transportation of the Asian arowana

One of the traditional enigmas in freshwater zoogeography has been the evolutionary origin of Scleropages formosus inhabiting Southeast Asia (the Asian arowana), which is a species threatened with extinction among the highly freshwater-adapted fishes from the order Osteoglossiformes. Dispersalists have hypothesized that it originated from the recent (the Miocene or later) transmarine dispersal of morphologically quite similar Australasian arowanas across Wallace's Line, but this hypothesis has been questioned due to their remarkable adaptation to freshwater. We determined the complete nucleotide sequences of two mitochondrial protein genes from 12 osteoglossiform species, including all members of the suborder Osteoglossoidei, with which robust molecular phylogeny was constructed and divergence times were estimated. In agreement with previous morphology-based phylogenetic studies, our molecular phylogeny suggested that the osteoglossiforms diverged from a basal position of the teleostean lineage, that heterotidines (the Nile arowana and the pirarucu) form a sister group of osteoglossines (arowanas in South America, Australasia, and Southeast Asia), and that the Asian arowana is more closely related to Australasian arowanas than to South American ones. However, molecular distances between the Asian and Australasian arowanas were much larger than expected from the fact that they are classified within the same genus. By using the molecular clock of bony fishes, tested for its good performance for rather deep divergences and calibrated using some reasonable assumptions, the divergence between the Asian and Australasian arowanas was estimated to date back to the early Cretaceous. Based on the molecular and geological evidence, we propose a new model whereby the Asian arowana vicariantly diverged from the Australasian arowanas in the eastern margin of Gondwanaland and migrated into Eurasia on the Indian subcontinent or smaller continental blocks. This study also implicates the relatively long absence of osteoglossiform fossil records from the Mesozoic.     

An approach to early genetic alterations in precancerous cells

To investigate the potential role of the PTEN tumor-suppressor gene in the carcinogenesis of ovarian endometrioid carcinoma and its related subtype, clear cell carcinoma, we examined 20 ovarian endometrioid carcinomas, 24 clear cell carcinomas and 34 solitary endometrial cysts of the ovary for LOH at 10q23.3 and point mutations of the PTEN gene, using a laser-assisted microdissection method. LOH was found in 8 of 19 ovarian endometrioid carcinomas (42.1%), 6 of 22 clear cell carcinomas (27.3%) and 13 of 23 solitary endometrial cysts (56.5%). Somatic mutations in the PTEN gene were identified in 4 of 20 ovarian endometrioid carcinomas (20.0%), 2 of 24 clear cell carcinomas (8.3%) and 7 of 34 solitary endometrial cysts (20.6%). In 5 endometrioid carcinomas with endometriosis, 3 displayed LOH events common to both the carcinoma and the endometriosis. In 7 clear cell carcinomas with endometriosis, 3 displayed LOH events common to both the carcinoma and the endometriosis. In no cases there were LOH events in the endometriosis only. These results indicate that inactivation of the PTEN gene is an early event in the development of both endometrioid and clear cell carcinoma of the ovary. A laser-assisted microdissection method enables us to collect target cells without contamination by non-tumor cells. We expect that this technique will be very useful for investigating genetic alterations in cancerous or precancerous lesions. Early genetic alterations in various precancerous cells detected by light microscopy can be readily identified by the tissue-microdissection method.     

Linear Diagonals‐Parameter Symmetry and Quasi‐Symmetry Models for Cumulative Probabilities in Square Contingency Tables with Ordered Categories

For the analysis of square contingency tables with ordered categories, Caussinus (1965) considered the quasi‐symmetry (QS) model, Goodman (1979) considered the diagonals‐parameter symmetry (DPS) model, and Agresti (1983) considered the linear diagonals‐parameter symmetry (LDPS) model. These models show the structures of symmetry for cell probabilities. Tomizawa (1993) proposed another DPS model which has a similar multiplicative form for cumulative probabilities that an observation will fall in row (column) category i or below and column (row) category j (>i) or above. This paper proposes another LDPS and QS models that have the corresponding similar multiplicative forms for cumulative probabilities instead of cell probabilities. Special cases of the proposed models include symmetry. Two kinds of unaided distance vision data and endometrial cancer data are analyzed using these models. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A structural basis for depolymerization of alginate by polysaccharide lyase family-7

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.     

Elucidation of a carotenoid biosynthesis gene cluster encoding a novel enzyme, 2,2'-beta-hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2'-beta-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2'-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.     

New complete mitochondrial DNA sequence of the lancelet Branchiostoma lanceolatum (Cephalochordata) and the identity of this species' sequences

Three mitochondrial (mt) genes were sequenced for two Atlantic lancelet species, Branchiostoma lanceolatum and B. floridae, to examine a serious discrepancy among previously published results of molecular studies: substantial sequence difference in a nuclear gene vs. virtual identity in the mt genome sequence. The results revealed that three mt genes of B. lanceolatum, collected from Helgoland in the North Sea and Naples in the Mediterranean, were quite diverged from those of B. floridae, collected from Tampa Bay, Florida. Therefore, the previously recognized identity in the mt genome between the two species is attributable to misidentification of materials used. To correct this misleading information, the complete mtDNA sequence of B. lanceolatum was determined for an individual from Helgoland.     

Identification of the critical region in replication factor C from Pyrococcus furiosus for the stable complex formation with proliferating cell nuclear antigen and DNA

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis. The function of RFC is to load PCNA, a processivity factor of replicative DNA polymerases, onto primed DNA templates. The central hole of the PCNA homo-trimeric ring encircles doublestranded DNA, so that DNA polymerases can operate for DNA synthesis with PCNA along a DNA template. The Pyrococcus furiosus RFC (PfuRFC) consists of a small subunit (RFCS, 37kDa) and a large subunit (RFCL, 55kDa), which show significant sequence identity to the eukaryotic homologs. The C-terminal region of RFCL has an acidic cluster of about 30 amino acids, which consists mainly of glutamic acid residues, and a following basic cluster of 10 amino acids, which consists mainly of lysine residues. These clusters of charged amino acids, which precede the C-terminal consensus sequence, PIP (PCNA interacting protein)-box, are conserved in several archaeal RFCLs. The series of mutant PfuRFC containing the C-terminal deletions in RFCL were constructed. The mutational analyses showed that the charged cluster is not essential for loading of PCNA onto DNA. However, the region containing the basic cluster is important for the stable ternary (RFC-PCNA-DNA) complex formation.     

Characterization of β -Carotene Ketolases, CrtW, from Marine Bacteria by Complementation Analysis in Escherichia coli

A complementation analysis was performed in Escherichia coli to evaluate the efficiency of β-carotene ketolases (CrtW) from the marine bacteria Brevundimonas sp. SD212, Paracoccus sp. PC1 (Alcaligenes PC-1), and Paracoccus sp. N81106 (Agrobacterium aurantiacum), for astaxanthin production. Each crtW gene was expressed in Escherichia coli synthesizing zeaxanthin due to the presence of plasmid pACCAR25ΔcrtX. Carotenoids that accumulated in the resulting E. coli transformants were examined by chromatographic and spectroscopic analyses. The transformant carrying the Paracoccus sp. PC1 or N81106 crtW gene accumulated high levels of adonixanthin, which is the final astaxanthin precursor for CrtW, and astaxanthin, while the E. coli transformant with crtW from Brevundimonas sp. SD212 did not accumulate any adonixanthin and produced a high level of astaxanthin. These results show efficient conversion by CrtW of Brevundimonas sp. SD212 from adonixanthin to astaxanthin, which is a new-found characteristic of a bacterial CrtW enzyme. The phylogenetic positions between CrtW of the two genera, Brevundimonas and Paracoccus, are distant, although they fall into α-Proteobacteria.     

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).     

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.