Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

Relationships between Micro-Vascular and Iodine-Staining Patterns in the Vicinity of the Tumor Front of Superficial Esophageal Squamous Carcinoma

Objective

The aim of the present study was to clarify differences between micro-vascular and iodine-staining patterns in the vicinity of the tumor fronts of superficial esophageal squamous cell carcinomas (ESCCs).

Methods

Ten consecutive patients with ESCCs who were treated by endoscopic submucosal dissection (ESD) were enrolled. At the edge of the iodine-unstained area, we observed 183 sites in total using image-enhanced magnifying endoscopy. We classified the micro-vascular and iodine-staining patterns into three types: Type A, in which the line of vascular change matched the border of the iodine-unstained area; Type B, in which the border of the iodine-unstained area extended beyond the line of vascular change; Type C, in which the line of vascular change extended beyond the border of the iodine-unstained area. Then, by examining histopathological sections, we compared the diameter of intra-papillary capillary loops (IPCLs) in cancerous areas and normal squamous epithelium.

Results

We investigated 160 sites that the adequate quality of pictures were obtained. There was no case in which the line of vascular change completely matched the whole circumference of the border of an iodine-unstained area. Among the 160 sites, type A was recognized at 76 sites (47.5%), type B at 79 sites (49.4%), and type C at 5 sites (3.1%). Histological examination showed that the mean diameter of the IPCLs in normal squamous epithelium was 16.2±3.7μm, whereas that of IPCLs in cancerous lesions was 21.0±4.4μm.

Conclusions

The development of iodine-unstained areas tends to precede any changes in the vascularity of the esophageal surface epithelium.     

Engineering issues of microbial ecology in space agriculture]

Closure of the materials recycle loop for water-foods-oxygen is the primary purpose of space agriculture on Mars and Moon. A microbial ecological system takes a part of agriculture to process our metabolic excreta and inedible biomass and convert them to nutrients and soil substrate for cultivating plants. If we extend the purpose of space agriculture to the creation and control of a healthy and pleasant living environment, we should realize that our human body should not be sterilized but exposed to the appropriate microbial environment. We are proposing a use of hyper-thermophilic aerobic composting microbial ecology in space agriculture. Japan has a broad historical and cultural background on this subject. There had been agriculture that drove a closed loop of materials between consuming cities and farming villages in vicinity. Recent environmental problems regarding garbage collection and processing in towns have motivated home electronics companies to innovate "garbage composting" machines with bacterial technology. Based on those matured technology, together with new insights on microbiology and microbial ecology, we have been developing a conceptual design of space agriculture on Moon and Mars. There are several issues to be answered in order to prove effectiveness of the use of microbial systems in space. 1) Can the recycled nutrients, processed by the hyper-thermal aerobic composting microbial ecology, be formed in the physical and chemical state or configuration, with which plants can uptake those nutrients? A possibility of removing any major components of fertilizer from its recycle loop is another item to be evaluated. 2) What are the merits of forming soil microbial ecology around the root system of plants? This might be the most crucial question. Recent researches exhibit various mutually beneficial relationships among soil microbiota and plants, and symbiotic ecology in composting bacteria. It is essential to understand those features, and define how to conduct preventive maintenance for keeping cultivating soil healthy and productive. 3) Does microbial ecology contribute to building sustainable and expandable human habitation by utilizing the on site extraterrestrial resources? We are assessing technical feasibility of converting regolith to farming soil and structural materials for space agriculture. In the case of Mars habitation, carbon dioxide and a trace amount of nitrogen in atmosphere, and potassium and phosphor in minerals are the sources we consider. Excess oxygen can be accumulated by woods cultivation and their use for lumber. 4) Is the operation of space agriculture robust and safe, if it adopts hyper-thermophilic aerobic microbial ecology? Any ecological system is complex and non-linear, and shows latency and memory effects in its response. It is highly important to understand those features to design and operate space agriculture without falling into the fatal failure. Assessment should be made on the microbial safety and preparation of the preventive measures to eliminate negative elements that would either retard agricultural production or harm the healthy environment. It is worth to mention that such space agriculture would be an effective engineering testbed to solve the global problem on energy and environment. Mars and Moon exploration itself is a good advocate of healthy curiosity expressed by the sustainable civilization of our humankind. We propose to work together towards Mars and Moon with microbial ecology to assure pleasant habitation there.     

Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N⁴-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N⁴-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N¹-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k_cat/K_m value for N¹-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N¹-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k_cat/K_m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N¹-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N¹-aminopropylagmatine. Furthermore, spermine and N⁴-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.Polyamines are positively charged aliphatic compounds. Putrescine [4], spermidine [34], and spermine [343] are common polyamines observed in various living organisms, from viruses to humans (16). Polyamines, which play important roles in cell proliferation and cell differentiation (19, 34), are thought to contribute to adaptation against various stresses (9, 26). In thermophilic microorganisms, polyamines contribute to growth under high-temperature conditions. Indeed, in the thermophilic bacterium Thermus thermophilus, a mutant strain lacking the enzyme related to polyamine biosynthesis shows defective growth at high temperatures (23). Furthermore, thermophilic archaea and bacteria possess long-chain and branched-chain polyamines such as N⁴-aminopropylspermidine [3(3)4], N⁴-aminopropylspermine [3(3)43], and N⁴-bis(aminopropyl)spermidine [3(3)(3)4], in addition to common polyamines (11, 13, 14). N⁴-aminopropylspermine was detected in the cells of thermophiles, such as Saccharococcus thermophilus, thermophilic Bacillus and Geobacillus spp. (Bacillus caldolyticus, B. caldotenax, B. smithii, Geobacillus stearothermophilus, and G. thermocatenulatus), Caldicellulosiruptor spp. (C. kristjanssonii and C. owensensis) and Calditerricola spp. (C. satsumensis and C. yamamurae) (10, 12, 22), but it was not detected in archaea. These unique polyamines are thought to support the growth of thermophilic microorganisms under high-temperature conditions. An in vitro study indicated that long-chain and branched-chain polyamines effectively stabilized DNA and RNA, respectively (32).Polyamines are synthesized from amino acids such as arginine, ornithine, and methionine (26). In most eukaryotes, putrescine is synthesized directly from ornithine by ornithine decarboxylase (34). Plants and some bacteria possess additional or alternative putrescine biosynthesis pathways in which putrescine is synthesized from arginine via agmatine (18, 31, 35). In this pathway, agmatine is synthesized by arginine decarboxylase, and agmatine is converted to putrescine by agmatine ureohydrolase or a combination of agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase. Longer polyamines are then produced by the addition of the aminopropyl group from decarboxylated S-adenosylmethionine. This pathway is shown on the left in Fig. ​Fig.11 (pathway I). On the other hand, the thermophilic bacterium T. thermophilus possesses a unique polyamine-biosynthetic pathway (23) in which spermidine is synthesized from agmatine via N¹-aminopropylagmatine by aminopropyl transferase followed by ureohydrolase, as shown on the right in Fig. ​Fig.11 (pathway II).Open in a separate windowFIG. 1.Predicted biosynthetic pathway of polyamines in T. kodakarensis. (A) Predicted biosynthetic pathway. Pyruvoyl-dependent arginine decarboxylase proenzyme (TK0149), arginine/agmatine ureohydrolases (TK0240/TK0474/TK0882), aminopropyl transferase (TK0147), and pyruvoyl-dependent S-adenosylmethionine decarboxylase proenzyme (TK1592) are shown based on the genome analysis. (B) Structures of unique polyamines.A sulfur-reducing hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, was isolated from Kodakara Island, Kagoshima, Japan (1, 21). This archaeon grows at temperatures between 60°C and 100°C but optimally at 85°C. Under low- or high-temperature-stressed conditions, T. kodakarensis produces cold- or heat-inducible chaperones to adapt to unfavorable growth environments (4, 5, 30). The lipid composition of the membrane also changes depending on the growth shift (20). In addition to acting as such tolerance factors, polyamines have been suggested to play an important role in maintaining nucleosomes in high-temperature environments (15). A complete genome analysis of T. kodakarensis has been performed, and the pathway of polyamine biosynthesis has been predicted (Fig. ​(Fig.1)1) (6, 7). It has been speculated that putrescine is synthesized from arginine via agmatine by arginine decarboxylase (PdaD_Tk) and agmatine ureohydrolase. Long- and/or branched-chain polyamines are then produced by the addition of the aminopropyl group derived from decarboxylated S-adenosylmethionine. Previously, we revealed that PdaD_Tk catalyzed the first step of polyamine biosynthesis and was essential for cell growth (6). The strain DAD, which lacks the gene pdaD_Tk, does not grow in medium without agmatine. Archaeal cells are known to use agmatine to synthesize agmatidine, which is an agmatine-conjugated cytidine found at the anticodon wobble position of archaeal tRNA^Ile (17). Agmatine is important for agmatidine synthesis as well as long-chain polyamine. In the present study, we focused on the subsequent steps in polyamine biosynthesis, especially from agmatine to spermidine. T. kodakarensis possesses three agmatine ureohydrolase homologues (TK0240, TK0474, and TK0882); however, it is unclear which one is dominantly functional in T. kodakarensis cells. In a closely related genus, Pyrococcus, TK0474 and TK0882 orthologues have been identified, but the TK0240 orthologue is missing in Pyrococcus genomes. In Pyrococcus horikoshii, PH0083, which is an orthologue of TK0882, was shown to possess agmatine ureohydrolase activity (8). TK0882, hence, appears to possess agmatine ureohydrolase activity as well. It is unclear whether other agmatine ureohydrolase homologues (TK0240 and TK0474) are involved in polyamine synthesis and cell growth in T. kodakarensis. In addition to agmatine ureohydrolase, aminopropyl transferase plays a crucial role in the synthesis of polyamines. TK0147 was annotated first as spermidine synthase and shares sequence identity with aminopropyl transferase (PF0127) from Pyrococcus furiosus (3). It is therefore expected to harbor the function of aminopropyl transferase for long-chain-polyamine synthesis. Recombinant PF0127 showed broad amine acceptor specificity for agmatine, 1,3-diaminopropane (3), putrescine, cadaverine (5), sym-nor-spermidine (33), and spermidine. While maximal catalytic activity was observed with cadaverine, agmatine was most often preferred on the basis of the k_cat/K_m value (3), suggesting that pathway II is a dominant route for polyamine synthesis in P. furiosus. In the present study, various disruptants lacking genes for polyamine biosynthesis were constructed in order to understand the physiological roles of these enzymes in T. kodakarensis. The cell growth profiles and cytoplasmic polyamines of the wild type and the disruptants were analyzed and compared. Recombinant enzymes were also purified and characterized. The obtained results are expected to provide useful information regarding the specific roles of polyamines in thermophiles.     

Archaeal pre-mRNA splicing: a connection to hetero-oligomeric splicing endonuclease

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.     

The plasmids found in isolates of the acidothermophilic archaebacterium Thermoplasma acidophilum

Abstract Plasmids were detected in isolates of an acidothermophilic archaebacterium Thermoplasma acidophilum . One of the plasmids, pTA1, was characterized. The plasmid was a circular DNA of 15.2 kbp. A physical map was constructed using three restriction endonucleases. A copy number of this plasmid was estimated to be 7–13 per cell. The homologous sequence was not found in the chromosomal DNA of the host cell.     

sn-Glycerol-1-Phosphate-Forming Activities in Archaea: Separation of Archaeal Phospholipid Biosynthesis and Glycerol Catabolism by Glycerophosphate Enantiomers

In Methanobacterium thermoautotrophicum, sn-glycerol-1-phosphate (G-1-P) dehydrogenase is responsible for the formation of the Archaea-specific backbone of phospholipids, G-1-P, from dihydroxyacetonephosphate (DHAP). The possible G-1-P-forming activities were surveyed in cell-free extracts of six species of Archaea. All the archaeal cell-free homogenates tested revealed the ability to form G-1-P from DHAP. In addition, activities of G-3-P-forming glycerol kinase and G-3-P dehydrogenase were also detected in four heterotrophic archaea, while glycerol kinase activity was not detected in two autotrophic methanogens. These results show that G-1-P is produced from DHAP by G-1-P dehydrogenase in a wide variety of archaea while exogenous glycerol is catabolized via G-3-P.     

Abiotic synthesis of amino acids and imidazole by proton irradiation of simulated primitive earth atmospheres

Proton irradiation of simulated primitive earth atmosphere was performed, and amino acids and imidazole were analyzed. A mixture of carbon monoxide and nitrogen over water was irradiated by high energy protons (3 MeV, 0.6 µA) generated by a Van de Graaff accelerator for 2–5 h. Various kinds of proteinous and non-proteinous amino acids were detected in the irradiation products. Imidazole present in the irradiation products was also detected by high-performance liquid chromatography and mass spectrometry. The present results suggest that compounds of biological importance such as amino acids could be synthesized from primitive earth atmosphere by radiation of cosmic rays and/or solar flare particles.     

Screening of stable proteins in an extreme thermophile, Thermus thermophilus

The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.