Metallosphaera sedula TA-2, a calditoglycerocaldarchaeol deletion strain of a thermoacidophilic archaeon.

A spherical thermoacidophilic archaeon, strain TA-2, was obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. This isolate is an obligate aerobic chemoorganoheterotroph that grows optimally at about 75 degrees C, pH 2.8. The G + C content of DNA from TA-2 is 47 mol%. The 16S rRNA gene from TA-2 showed more than 99% similarity with those of Metallosphaera sedula and Metallosphaera prunae and less than 92% similarity with other members of the order Sulfolobales. DNA-DNA hybridization experiments showed more than 93% genomic DNA homology among TA-2, M. sedula DSM5348T, and M. prunae DSM10039T. However, TA-2 lacks calditoglycerocaldarchaeol derivatives, which are usually found in the membrane lipids of members of the order Sulfolobales. Therefore, calditoglycerocaldarchaeol may not be essential for survival in thermophilic and acidophilic environments. The isolate was deposited as Metallosphaera sedula TA-2 (JCM 9064, IFO 15160).

Variation in molecular species of polar lipids from thermoplasma acidophilum depends on growth temperature.

Five types of molecular species of C40 isoprenoid chains, having different numbers of cyclopentane rings, were detected in the ether core lipid of Thermoplasma acidophilum. The average cyclization number of the hydrocarbon chains in the lipids increased with increasing growth temperatures.

The evolution of lipids.

Living organisms on the Earth which are divided into three major domains--Archaea, Bacteria, and Eucarya, probably came from a common ancestral cell. Because there are many thermophilic microorganisms near the root of the universal phylogenetic tree, the common ancestral cell should be considered to be a thermophilic microorganism. The existence of a cell is necessary for the living organisms; the cell membrane is the essential structural component of a cell, so its amphiphilic property is vital for the molecule of lipids for cell membranes. Tetraether type glycerophospholipids with C40 isoprenoid chains are major membrane lipids widely distributed in archaeal cells. Cyclization number of C40 isoprenoid chains in thermophilic archaea influences the fluidity of lipids whereas the number of carbons and degree of unsaturation in fatty acids do so in bacteria and eucarya. In addition to the cyclization of the tetraether lipids, covalent bonding of two C40 isoprenoid chains was found in hyperthermophiles. These characteristic structures of the lipids seem to contribute to their fundamental physiological roles in hyperthermophiles. Stereochemical differences between G-1-P archaeal lipids and G-3-P bacterial and eucaryal lipids might have occurred by the function of some proteins long after the first cell was developed by the reactions of small organic molecules. We propose that the structure of lipids of the common ancestral cell may have been similar to those of hyperthermophilic archaea.

Characterization of caldarchaetidylglycerol analogs, dialkyl-type and trialkyl-type, from Thermoplasma acidophilum.

The structures of three kinds of phospholipids (PL-X, PL-Y, and PL-T) isolated from Thermoplasma acidophilum have been characterized. The core lipid of PL-Y was caldarchaeol, and that of PL-X was archaeol. The composition of the hydrocarbon chains of the PL-T core lipid was C20 phytane and C40 isoprenoid in a molar ratio of 2 to 1. The major molecular species of the C40 isoprenoid was acyclic without the cyclopentane ring. These three kinds of intact phospholipids commonly had glycerophosphate residues as polar head groups. The structure of PL-T was characterized as trialkyl-type caldarchaetidylglycerol, PL-Y as caldarchaetidylglycerol, and PL-X as archaetidylglycerol.

Sequence analysis of three family B DNA polymerases from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis.

Three family B DNA polymerase genes, designated B1, B2, and B3, were cloned from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis, and sequenced. Deduced amino acid sequences of B1 and B3 DNA polymerases have all exonuclease and polymerase motifs which include critical residues for catalytic activities. Furthermore, a YxGG/A motif, which is located between 3'-5' exonuclease and polymerization domains of family B DNA polymerases, was also found in each of the B1 and B3 sequences. These findings suggested that S. ohwakuensis B1 and B3 DNA polymerases have both exonuclease and polymerase activities. However, amino acid sequence of the B2 DNA polymerase of this organism contains several amino acid substitutions in Pol-motifs, and also lacks Exo-motif I and Exo-motif II. These substitutions and lack of certain motifs raise questions about polymerase and exonuclease activities of the corresponding gene product. The B3 sequence of S. ohwakuensis is more closely related to Pyrodictium, Aeropyrum, and Archaeoglobus DNA polymerase B3 sequences than to the Sulfolobus B3 sequences. Phylogenetic analysis showed that crenarchaeal B1 DNA polymerases are closely related to each other, and suggested that crenarchaeal B3, euryarchaeal family B, and eukaryal epsilon DNA polymerases may be orthologs.

Glucosylcaldarchaetidylglycerol, a minor phosphoglycolipid from Thermoplasma acidophilum.

A novel phosphoglycolipid (GPL-K) was isolated from Thermoplasma acidophilum (ATCC 27658). The chemical components of GPL-K were analyzed by gas liquid chromatography and GC-MS. The sugar moiety of GPL-K and its anomeric region were analyzed by NMR assignment. The core lipid of GPL-K was caldarchaeol, and its main hydrocarbon chains were acyclic and monocyclic C(40) biphytanyl. The polar head groups were alpha-glucose and glycerophosphate. The negative FAB-MS spectrum of GPL-K confirmed that the lipid peak of m/z 1614 consists of a caldarchaeol (including one cyclopentane ring), a hexose sugar, and a glycerophosphate. We have proposed the tentative structure of GPL-K.

Pre-treatment with mild whole-body heating prevents gastric ulcer induced by restraint and water-immersion stress in rats.

The purpose of this study was to assess the preventive effect of pre-mild whole-body heating (WBH) on gastric ulcer induced by restraint and water-immersion stress. The ulcer index and ulcer area ratio in rats exposed to restraint and water-immersion stress were significantly decreased (p < 0.05 for both) after pre-treatment with mild WBH, compared with non-pre-treated rats. Mortality of rats among the pre-treated with mild WBH (0%) was lower than in the control group (33%). The concentration of HSP 70f in the stomach (both fundic and pyloric mucosal areas) of rats pre-treated with mild WBH was significantly higher than in animals exposed to restraint and water-immersion stress alone (p < 0.05) before exposed to stress, but was not significantly higher immediately after stress or 1 or 3 days later. The HSP 70f content of peripheral lymphocytes was increased by the pre-treatment with mild WBH. These results suggest that HSP 70f induced by pre-treatment with mild hyperthermia protects against more severe stress due to restraint and water-immersion, thereby preventing gastric ulcer formation. Pre-treatment with mild WBH is the safest cytoprotective method through the accumulation of HSP 70f. The concentration of HSP 70f in peripheral lymphocytes may be a useful clinical laboratory indicator for assessing the level of HSP 70f as having cytoprotective activity.

Phylogenetic analysis of archaeal PCNA homologues.

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27kDa) and 248 aa (27kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages.

Partial purification and characterization of thermostable acid phosphatase from thermoacidophilic archaeon Sulfolobus acidocaldarius.

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70 degrees C, respectively. The apparent K(m) value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75 degrees C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein.

Isolation and characterization of novel neutral glycolipids from Thermoplasma acidophilum.

Several novel neutral glycolipids (GL-1a, GL-1b, GL-2a, GL-2b and GL-2c) were isolated from Thermoplasma acidophilum by high-performance liquid chromatography using phenylboronic acid-silica and preparative thin-layer chromatography. The tentative structures of these lipids were characterized by the combination of gas-liquid chromatography, the methylation procedure, and (1)H-NMR and FAB-mass spectrometries. The lipophilic portion of the neutral glycolipids was composed of a simple molecular species named caldarchaeol (dibiphytanyl-diglycerol tetraether). The sugar moieties of these glycolipids were composed of gulose and glucose which formed monosaccharide residues on one side or both sides of the core lipids. Gulose was attached to the terminal glycerol OH group of the core lipid with a beta-configuration and glucose being attached with an alpha-configuration. The proposed structure of GL-1a was gulosylcaldarchaeol and that of GL-1b was glucosylcaldarchaeol. The structures of GL-2a, GL-2b, and GL-2c were the analogs of the caldarchaeol derivatives attached by a variety of gulosyl residues or glucosyl residues on both sides of the terminal OH groups.

Sulfurisphaera ohwakuensis gen. nov., sp. nov., a novel extremely thermophilic acidophile of the order Sulfolobales.

Three spherical thermoacidophilic archaea (strains TA-1T, TA-13, TA-14) were obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. All the isolates are facultatively anaerobic, and grew optimally at around 85 degrees C, pH 2.0. Isolate TA-1T was characterized further. The G + C content of DNA from TA-1T is 33 mol%. Although these properties resemble those of the genus Acidianus, the sequence of the 16S rRNA gene from strain TA-1T was more similar to that of species of Stygiolobus than of Acidianus. DNA-DNA hybridization experiments also indicated that strain TA-1T is clearly distinguished phylogenetically from the members of Acidianus, Sulfolobus and Metallosphaera. On the basis of the distinct physiological and molecular properties, we describe the new strains as members of the new genus Sulfurisphaera. The type species of the genus is Sulfurisphaera ohwakuensis, and the type strain of the species is TA-1T (= IFO 15161T).

[Increase of cellular fibrinolysis in human lung cancer cell line by radiation: relationship between urokinase-type plasminogen activator (uPA) and metastasis and invasion].

It is well known that urokinase-type plasminogen activator (uPA) activates fibrinolysis of tumor cells and accelerates their metastasis and invasion. Human adenosquamous cell line, AOI cells, were stimulated to produce and accumulate of uPA by radiation. In AOI cells, there was relationship between uPA production and accumulation and the radiation doses. It was suggested that radiation had the possibility to accelerate the metastasis and invasion by increasing the production and accumulation of uPA from cancer cells.

[A new quantitative assay of heat shock protein 70 by based on an enzyme-linked immunosorbent assay method].

Heat Shock Protein 70 (HSP70) was very important stress protein. HSP70 had been detected by immunoblotting or mRNA of HSP70. Our purpose is to develop an easy quantitative assay of HSP70 and utilize clinical samples, cells, tissues etc. We examined a new quantitative assay of HSP70 that was based on an enzyme-linked immunosorbent assay (ELISA). This quantitative assay showed reproducible standard curve, high reproducibility (CV : 7.1, 6.8%) and recovery (93 approximately 116%). The determination of HSP70 in human umbilical vein endothelial cells (HUVEC) by heat stress was assayed using this method. HSP70 in HUVEC increased according to the heat temperature. It is concluded that the quantity of HSP70 might be possible even in cells and tissues by this method.

[Proton-magnetic resonance spectroscopy (1H-MRS) of human plasma obtained from patients with various cancer--new detection method for common cancer].

1H-Magnetic Resonance (MR) Spectra of plasma obtained from patients with various cancer were measured. Clinical usefulness of 1H-MRS assay for detection of common cancer was examined. The following results were obtained. 1. High lactate peak was detected significantly with 1H-MR Spectra of plasma in every cancer patients. The patterns of 1H-MR spectra were very similar in each case, except for the lactate peak. 2. Lactate values (area ratio of lactate peak to TSP) of cancer patients showed high correlation with tumor marker of AFP, but not significant with CEA and CA19-9. 3. 1H-MRS assay has many advantages in the clinical examination. From these results, it was concluded that lactate peak in 1H-MRS might be useful to detect common cancer as a new tumor marker and 1H-MRS assay was equipped with enough characters as a new clinical laboratory assay for detection of specific metabolic deficiency.

The structure of the core polyol of the ether lipids from Sulfolobus acidocaldarius.

The major ether-type lipid structures of Sulfolobus acidocaldarius (ATCC33909) were composed of caldarchaeol and calditoglycerocaldarchaeol. However, the characterization by nuclear magnetic resonance spectroscopy and mass spectrometry showed that the structure of calditol in calditoglycerocaldarchaeol is not nonitol, 2-(1',2',3'-trihydroxypropyl)1,2,3,4,5,6-hexahydroxyhexane, but 2-hydroxymethyl-1-(2,3-dihydroxypropoxy)2,3,4,5-cyclopentanetet raol with an ether linkage in the molecule. Such an intermolecular ether linkage was resistant to BCl3 treatment, but nonresistant to 57% HI degradation treatment conducted at 100 degrees C for 60 h, producing 2-hydroxymethyl-1,2,3,4,5-cyclopentanepentaol from calditol as reaction product. Further, it was confirmed that the structure of calditol is essentially a derivative of glycerol, and hydrocarbon chains were conjugated to the glycerol-like site in the structure. The calditol with an ether linkage in the molecule suggested an important role regarding the properties of heat-resistance and acid-resistance observed in Sulfolobales.

[19F-MR imaging of transplanted tumor: perfluorochemicals as a fluorine tumor imaging agent].

PURPOSE: The purpose of our study was to detect tumor selectively using 19F-magnetic resonance imaging (MRI) and to assess Fluosol-DA, a perfluorochemical emulsion, as a tumor imaging agent for 19F-MRI. MATERIALS AND METHODS: SCC VII cells were transplanted in the right leg of mice. After 6 days, Fluosol-DA was administrated intravenously (40 ml/kg). 19F-MR imaging was performed on a Bruker CSI Omega 2 at 4.7 Tesla using a homemade volume coil. RESULTS: In vitro, the concentration and 19F signal intensity of FDA showed a very high correlation (r = 0.9997). Detection on MRI was possible at a concentration of 2%. In vivo, images of 19F in SCC VII tumors were achieved in animals 2 days after the administration of FDA. CONCLUSION: Our study confirms the feasibility of 19F-MR in vivo imaging of tumors using the fluorine compound FDA.

[Thermotolerance and heat shock protein (HSP 70) in vascular endothelial cells].

Recently, it was determined that the endothelial cells of blood vessel play a very important physiological role in the regulation of blood coagulation and selective permeability. To study the thermotolerance of vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were heated at 40, 43, 45 or 50 degrees C for various lengths of time with or without preheating at 40 or 43 degrees C for 30 min. The cell viability (CV) of HUVEC decreased gradually according to heating time. However, the CV of preheated HUVEC decreased slightly or not at all. Heat shock protein (HSP) in HUVEC heated at 37, 40, 43, or 45 degrees C was examined by immunoblotting. A new HSP 70 band was detected in HUVEC by heating at 40, 43 or 45 degrees C. HUVEC revealed thermotolerance with induction of HSP by heat stress.