Plant glycerol-3-phosphate-1-acyltransferase (GPAT): structure selectivity studies

Squash glycerol-3-phosphate-1-acyltransferase has been crystallized and the structure of the enzyme determined, at 1.9-A resolution, using multiple isomorphous replacement of the wild type and a series of individual cysteine mutants. Competitive in vitro substrate selectivity assays have been established that differentiate between selective and non-selective forms of the enzyme. Particular care was taken to use near-physiological concentrations of both substrates. Clear substrate selectivity can be demonstrated with the natural substrate acyl-acyl carrier protein but not with the substrate analogue acyl-CoA. The use of site-directed mutagenesis, coupled to three-dimensional structural determinations, should provide a rational basis for elucidating structural components important in determining the substrate selectivity of this enzyme.     

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.     

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)     

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.     

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.     

Genetic differentiation between two color morphs of <Emphasis Type="Italic">Apogon taeniophorus</Emphasis> from southern Japan

The anterior half of the mitochondrial 16S rRNA gene (ca. 610bp) was compared for two color morphs (spotted and lined types) of a dark-striped cardinalfish, previously identified as Apogon taeniophorus. Phylogenetic analyses using maximum-parsimony (MP) and neighbor-joining (NJ) methods, with haplotypes of A. cookii as an outgroup, showed that the haplotypes of each color-morph were reciprocally monophyletic with 100% bootstrap values. In addition, the degree of sequence difference between the two morphs was comparable to that existing between the other clearly distinct congeneric species. These results, together with the differences in coloration and overlapped geographical ranges, indicated that the two color morphs of A. taeniophorus represent two distinct species.     

Characterization of a novel mammalian SUMO-1/Smt3-specific isopeptidase, a homologue of rat axam, which is an axin-binding protein promoting beta-catenin degradation

A novel SUMO-1/Smt3-specific isopeptidase, SMT3IP2/Axam2 (Smt3-specific isopeptidase 2), was cloned and characterized. The catalytic domains in the carboxyl-terminal region were very much similar to those of other SUMO-1/Smt3-specific proteases, but the amino-terminal part was quite different. The enzyme specifically bound to Smt3a and Smt3b but not to SUMO-1. The SMT3IP2 expressed by Escherichia coli could cleave SUMO-1, Smt3a, or Smt3b from a SUMO-1/RanGAP1, Smt3a/RanGAP1, or Smt3b/RanGAP1 conjugate, respectively, and had the activity of a carboxyl-terminal hydrolase to produce a glycine residue in the carboxyl terminus of these ubiquitin-like proteins. The sequence data indicated that the amino acid sequence of SMT3IP2 was mostly identical to that of rat Axam, which binds to Axin and promotes the degradation of beta-catenin, although its amino-terminal region was much shorter than that of Axam. Therefore, we designated this isopeptidase SMT3IP2/Axam2. When human SW480 cells were transfected with wild-type SMT3IP2/Axam2, the beta-catenin disappeared. When the cells were transfected with the SMT3IP2/Axam2 C500A mutant, which had neither isopeptidase nor carboxyl-terminal hydrolase activity, or with the 1-352 mutant, which lacked the catalytic domain of the enzyme, again the beta-catenin disappeared, indicating that the enzyme activities were not necessary for the instability of beta-catenin in this transfection assay system and that its competition with Dvl for binding to Axin may be important for the instability of beta-catenin as suggested previously for Axam (Kadoya, T., Kishida, S., Fukui, A., Hinoi, T., Michiue, T., Asashima, M., and Kikuchi, A. (2000) J. Biol. Chem. 275, 37030-37037). The involvement of its enzyme activities in the Wnt signaling pathway remains to be elucidated.