Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.     

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases

Sphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent-free conditions. Bacterial and plasmodial neutral SMases also showed SPC-PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen-bond donor at the C-2 or sn-2 position in the substrate is required for recognition by the enzymes.     

Cytosolic r{beta}-Cyanoalanine Synthase Activity Attributed to Cysteine Synthases in Cocklebur Seeds. Purification and Characterization of Cytosolic Cysteine Synthases

The activity of rß-cyanoalanine synthase (CAS, EC4.4.1.9 [EC] ) in cotyledons of cocklebur seeds (Xanthium penn-sylvanicumWallr.) was detected both in the soluble and particulate fractions.The CAS activity of the soluble fraction (cytosolic CAS activity)was 10 times higher than that of the particulate fraction. TheCAS activity of the particulate fraction was confirmed to belocalized in the mitochondria. Both enzymatic activities wereclearly separated by non-denaturing PAGE. The enzyme with cytosolicCAS activity has been extensively purified and separated intothree different forms designated as cyt-1, cyt-2, and cyt-3.According to the SDS-PAGE analysis, the three enzymes are estimatedto be a homodimer composed of 35-kDa sub-units. The purifiedenzymes showed CS activity. Partial amino acid sequences ofcyt-1 were determined and had a high homology with cysteinesynthases (CS, EC 4.2.99.8 [EC] ) from other plant sources. The catalyticaction of the purified CSs in converting cyanide and cysteineinto H₂S and rß-cyanoalanine was confirmed by thedetection of significant ¹⁴CN incorporation into rß-cyanoalanine.These results indicated that cytosolic CAS activity is due tocytosolic CS and suggested that the CAS activity of CS is likelyto be involved in cyanide metabolism in plant tissues. (Received January 7, 1998; Accepted March 16, 1998)     

Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Developmental changes in crossover frequency in Arabidopsis

Parental genomes are generally rearranged by two processes during meiosis: one is the segregation of homologous chromosomes and the other is crossing over between such chromosomes. Although the mechanisms underlying chromosome segregation and crossing over are well understood because of numerous genetic and molecular investigations, their contributions to the rearrangement of genetic information have not yet been analysed at a genome-wide level in Arabidopsis thaliana. We established 343 CAPS or SSLP markers to identify polymorphisms between two different Arabidopsis ecotypes, Col and Ler, which are distributed at an average distance of approximately 400kb between pairs of markers throughout the entire genome. Using these markers, crossover frequencies and chromosome segregation were quantified with respect to sex and age. Our large-scale analysis demonstrated that: (i) crossover frequencies during pollen formation were 1.79 and 1.37 times higher than those during megaspore formation in early and late flowers, respectively (P<0.001); (ii) the crossover frequencies during pollen formation were not significantly different between early and late flowers of main shoots (P>0.05), whereas the frequencies increased 1.30 times with shoot age during megaspore formation (P<0.001); (iii) the effect of aging depended on the developmental age of the individual shoot rather than on the age of the whole plant; and (iv) five chromosomes were randomly selected and mixed during meiosis.     

Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus sp.

Enzymatic decomposition of gelatin layers on X-ray films and repeated utilization of enzyme for potential industrialization were investigated using thermostable alkaline protease from the alkaliphilic Bacillus sp. B21-2. The decomposition of gelatin layers at 50 °C with the mutant enzyme (Ala187 was replaced by Pro) was higher than those of the wild-type and other mutant enzymes. In the repeated experiment for every 60 min (20 U ml^–1, 50 °C), the mutant enzyme could be satisfactorily used five times while three times for the wild-type enzyme.     

Plant regeneration via direct and indirect adventitious shoot formation and chromosome-doubled somaclonal variation in Titanotrichum oldhamii (Hemsl.) Solereder

The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing 0.1 mg l⁻¹ benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing 0.1 mg l⁻¹ indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.     

Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase.

The crystal structure of O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) of hyperthermophilic archaeon Pyrococcuskodakaraensis strain KOD1 (Pk -MGMT) was determined by single isomorphous replacement method with anomalous scattering (SIRAS) at 1.8 A resolution. The archaeal protein is extremely thermostable and repairs alkylated DNA by suicidal alkyl transfer from guanine O6 to its own cysteine residue. Archaea constitute the third primary kingdom of living organisms, sharing characteristics with procaryotic and eucaryotic cells. They live in various extreme environments and are thought to include the most ancient organisms on the earth. Structural studies on hyperthermophilic archaeal proteins reveal the structural features essential for stability under the extreme environments in which these organisms live, and will provide the structural basis required for stabilizing various mesophilic proteins for industrial applications. Here, we report the crystal structure of Pk-MGMT and structural comparison of Pk-MGMT and methyltransferase homologue from Escherichia coli (AdaC, C-terminal fragment of Ada protein). Analyses of solvent-accessible surface area (SASA) reveals a large discrepancy between Pk-MGMT and AdaC with respect to the property of the ASA. In the Pk-MGMT structure, the intra-helix ion-pairs contribute to reinforce stability of alpha-helices. The inter-helix ion-pairs exist in the interior of Pk-MGMT and stabilize internal packing of tertiary structure. Furthermore, structural features of helix cappings, intra and inter-helix ion-pairs are found around the active-site structure in Pk-MGMT.     

Seasonal changes in stream salmonid population densities in two tributaries of a boreal river in northern Japan

We examined seasonal changes in population densities of stream salmonids (masu salmon Oncorhynchus masou, white-spotted charr Salvelinus leucomaenis, and rainbow trout O. mykiss) in two tributaries of the Shoro River, eastern Hokkaido, Japan. In one small tributary, water temperature was relatively high during the winter, and populations of salmon and trout increased through immigration at this time of the year, becoming dominant components of the salmonid assemblage; the density of charr in this stream decreased during the winter, but charr was dominant during the summer. In another medium-sized tributary, the water temperature fell to close to 0°C during the winter, and densities of salmon and charr decreased in this season, through emigration; trout were very rare in this stream. Seasonal patterns of stream salmonid densities vary among species and between localities, resulting in seasonal changes in species composition. For a comprehensive understanding of population processes, a whole-river survey across seasons will be necessary.