Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.     

Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat

Ca(2+), which enters cardiac myocytes through voltage-dependent Ca(2+) channels during excitation, is extruded from myocytes primarily by the Na(+)/Ca(2+) exchanger (NCX1) during relaxation. The increase in intracellular Ca(2+) concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na(+)/Ca(2+) exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na(+)/Ca(2+) exchange activity was detected in null mutant hearts. The Na(+)-dependent Ca(2+) exchange activity as well as protein content of NCX1 were decreased by approximately 50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na(+)-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na(+)-dependent Ca(2+) handling in the heart and aorta.     

MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts

At the onset of mitosis, the Golgi apparatus, which consists of several cisternae, disperses throughout the cell to be partitioned into daughter cells. The molecular mechanisms of this process are now beginning to be understood. To investigate the biochemical requirements and kinetics of mitotic Golgi membrane dynamics in polarized cells, we have reconstituted the disassembly of the Golgi apparatus by introducing Xenopus egg extracts into permeabilized Mardin-Darby canine kidney (MDCK) cells. We used green fluorescence protein (GFP)-tagged galactosyltransferase-expressing MDCK cells to analyze the morphological changes of the Golgi membrane in the semi-intact system. Analyses by fluorescence and electron microscopies showed that the Golgi disassembly can be dissected into two elementary processes morphologically. In the first process, the perinuclear Golgi stacks break into punctate structures, intermediates, which are comprised of mini-stacks of cisternae associating with apical microtubule networks. In the second process, the structures fragment more thoroughly or substantially relocate to the ER. Our analyses further showed that cdc2 kinase and mitogen-activated protein kinase kinase (MAPKK = MEK) are differently involved in these two processes: the first process is mainly regulated by MEK and the second mainly by cdc2.     

The primary electron acceptor of green sulfur bacteria,bacteriochlorophyll 663, is chlorophyll a esterified with Δ 2,6-phytadienol

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), ¹H- and ¹³C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7¹ signal was observed at the high-field side of the singlet P3¹ signal in BChl 663, while a doublet peak of P7¹ overlapped that of P11¹ in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13²-epimer of BChl a, BChl a′, were found to be present per reaction center, which may constitute the primary electron donor. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.