Dietary salt with nitric oxide deficiency induces nocturnal polyuria in mice via hyperactivation of intrarenal angiotensin II-SPAK-NCC pathway.

Nocturnal polyuria is the most frequent cause of nocturia, a common disease associated with a compromised quality of life and increased mortality. Its pathogenesis is complex, and the detailed underlying mechanism remains unknown. Herein, we report that concomitant intake of a high-salt diet and reduced nitric oxide (NO) production achieved through Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) administration in mice resulted in nocturnal polyuria recapitulating the clinical features in humans. High salt intake under reduced NO production overactivated the angiotensin II-SPAK (STE20/SPS1-related proline-alanine-rich protein kinase)-NCC (sodium chloride co-transporter) pathway in the kidney, resulting in the insufficient excretion of sodium during the day and its excessive excretion at night. Excessive Na excretion at night in turn leads to nocturnal polyuria due to osmotic diuresis. Our study identified a central role for the intrarenal angiotensin II-SPAK-NCC pathway in the pathophysiology of nocturnal polyuria, highlighting its potential as a promising therapeutic target.

Estimation of the contribution of short-lived radioiodines to the thyroid dose for the public in case of inhalation intake following the Fukushima accident.

The purpose of this paper is to present (1) the method of assessing the contribution of short-lived radioiodines to the thyroid for members of the public in Fukushima and neighbouring prefectures based on available data and (2) the results of a realistic assessment of such a contribution. The estimates of that contribution for the inhalation intake that occurred on the day of the main fallout (15 March 2011) are within 15 % of the dose to the thyroid from (131)I. The contribution to the thyroid dose from intake of (132)Te is higher than that from the intake of (133)I by a factor of ∼3. The contribution of short-lived radioiodines to the thyroid dose for the public in the case of inhalation intake occurring as early as March 12 might be as great as 30-40 %.

[Comparative analysis of the radionuclide composition in fallout after the Chernobyl and the Fukushima accidents].

The nuclear accident occurred at Fukushima Dai-ichi Nuclear Power Plant (NPP) (March 11, 2011) similarly to the accident at the Chernobyl NPP (April 26, 1986) is related to the level 7 of the INES. It is of interest to make an analysis of the radionuclide composition of the fallout following the both accidents. The results of the spectrometric measurements were used in that comparative analysis. Two areas following the Chernobyl accident were considered: (1) the near zone of the fallout - the Belarusian part of the central spot extended up to 60 km around the Chernobyl NPS and (2) the far zone of the fallout--the "Gomel-Mogilev" spot centered 200 km to the north-northeast of the damaged reactor. In the case of Fukushima accident the near zone up to about 60 km considered. The comparative analysis has been done with respect to refractory radionuclides (95Zr, 95Nb, 141Ce, 144Ce), as well as to the intermediate and volatile radionuclides 103Ru, 106Ru, 131I, 134Cs, 137Cs, 140La, 140Ba and the results of such a comparison have been discussed. With respect to exposure to the public the most important radionuclides are 131I and 137Cs. For the both accidents the ratios of 131I/137Cs in the considered soil samples are in the similar ranges: (3-50) for the Chernobyl samples and (5-70) for the Fukushima samples. Similarly to the Chernobyl accident a clear tendency that the ratio of 131I/137Cs in the fallout decreases with the increase of the ground deposition density of 137Cs within the trace related to a radioactive cloud has been identified for the Fukushima accident. It looks like this is a universal tendency for the ratio of 131I/137Cs versus the 137Cs ground deposition density in the fallout along the trace of a radioactive cloud as a result of a heavy accident at the NPP with radionuclides releases into the environment. This tendency is important for an objective reconstruction of 131I fallout based on the results of 137Cs measurements of soil samples carried out at late dates after the Fukushima accident.

Measurement of soil contamination by radionuclides due to the Fukushima Dai-ichi Nuclear Power Plant accident and associated estimated cumulative external dose estimation.

Soil sampling was carried out at an early stage of the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident. Samples were taken from areas around FDNPP, at four locations northwest of FDNPP, at four schools and in four cities, including Fukushima City. Radioactive contaminants in soil samples were identified and measured by using a Ge detector and included (129 m)Te, (129)Te, (131)I, (132)Te, (132)I, (134)Cs, (136)Cs, (137)Cs, (140)Ba and (140)La. The highest soil depositions were measured to the northwest of FDNPP. From this soil deposition data, variations in dose rates over time and the cumulative external doses at the locations for 3 months and 1y after deposition were estimated. At locations northwest of FDNPP, the external dose rate at 3 months after deposition was 4.8-98 µSv/h and the cumulative dose for 1 y was 51 to 1.0 × 10(3)mSv; the highest values were at Futaba Yamada. At the four schools, which were used as evacuation shelters, and in the four urban cities, the external dose rate at 3 months after deposition ranged from 0.03 to 3.8µSv/h and the cumulative doses for 1 y ranged from 3 to 40 mSv. The cumulative dose at Fukushima Niihama Park was estimated as the highest in the four cities. The estimated external dose rates and cumulative doses show that careful countermeasures and remediation will be needed as a result of the accident, and detailed measurements of radionuclide deposition densities in soil will be important input data to conduct these activities.

Amendments to (63)Ni production calculation for Hiroshima by Takamiya et al. and DS02 fluence data by Egbert et al.

In a previous paper, Takamiya et al. calculated (63)Ni production in copper samples exposed to the Hiroshima atomic bomb. More specifically, they used their experimental cross-section values of the (63)Cu(n,p)(63)Ni reaction and compared the result with that of the corresponding calculation in the radiation dosimetry system DS02, which used another set of cross-section values. These results were different, and the following two reasons were found: typographical errors in several energy boundary values in the DS02 report that was also used in the calculation by Takamiya et al. and an inappropriate assumption on the cross-section values of the low neutron energy region in the calculation by Takamiya et al. These two issues are described and amended in the present report.

Feasibility of using 236U to reconstruct close-in fallout deposition from the Hiroshima atomic bomb.

The first results on the feasibility of using (236)U to reconstruct the level and spatial distribution of close-in fallout deposition from the Hiroshima A-bomb are reported, coupled with the use of global fallout (137)Cs and (239+240)Pu. The results for global fallout (236)U in soil samples (0-30cm) from Ishikawa prefecture showed that the deposition density of (236)U from the global fallout can be accurately evaluated using AMS. All deposited (236)U, (137)Cs and (239+240)Pu appeared to have been recovered using 30-cm cores. It was also noted from the depth profiles for (236)U/(239+240)Pu and (236)U/(137)Cs ratios that the downward behavior of (236)U in the soil was apparently similar to that of (239+240)Pu, while the (137)Cs was liable to be retained in upper layers compared with (236)U and (239+240)Pu. The accumulated levels were 1.78×10(13)atomsm(-2) for (236)U, 4340Bqm(-2) for (137)Cs and 141Bqm(-2) for (239+240)Pu. The ratios of (236)U/(137)Cs and (236)U/(239+240)Pu were (4.10±0.12)×10(9) and (1.26±0.04)×10(11)atomsBq(-1), respectively. Results of (236)U, (137)Cs and (239+240)Pu measurements for the seven soil cores (0-30cm) from Hiroshima were discussed on the basis of ratios of (236)U/(137)Cs and (236)U/(239+240)Pu by comparing with those from the background area in Ishikawa, indicating that the global fallout dominates the current level of (236)U accumulation in soil in the Black-rain area around Hiroshima after the Hiroshima bomb, and the contribution of the close-in fallout (236)U produced by the Hiroshima A-bomb seems difficult to observe.

Fast neutrons measured in copper from the Hiroshima atomic bomb dome.

The first measurements of (63)Ni produced by A-bomb fast neutrons (above approximately 1 MeV) in copper samples from Hiroshima encompassed distances from approximately 380 to 5062 m from the hypocenter (the point on the ground directly under the bomb). They included the region of interest to survivor studies (approximately 900 to 1500 m) and provided the first direct validation of fast neutrons in that range. However, a significant measurement gap remained between the hypocenter and 380 m. Measurements close to the hypocenter are important as a high-value anchor for the slope of the curve for neutron activation as a function of distance. Here we report measurements of (63)Ni in copper samples from the historic Hiroshima Atomic Bomb Dome, which is located approximately 150 m from the hypocenter. These measurements extend the range of our previously published data for (63)Ni providing a more comprehensive and consistent A-bomb activation curve. The results are also in good agreement with calculations based on the current dosimetry system (DS02) and give further experimental support to the accuracy of this system that forms the basis for radiation risk estimates worldwide.

Baicalein 5,6,7-trimethyl ether activates peroxisomal but not mitochondrial fatty acid beta-oxidation.

Recently, we reported that baicalein 5,6,7-trimethyl ether (BTM), a flavonoid, is capable of activating fatty acid beta-oxidation in X-linked adrenoleukodystrophy (X-ALD) fibroblasts (FEBS Lett. 2005; 579: 409-414). The objective of this study was to clarify whether BTM activates peroxisomal and/or mitochondrial fatty acid beta-oxidation. We first analysed the effect of BTM on fatty acid beta-oxidation in fibroblasts derived from healthy controls as well as patients with X-ALD, mitochondrial carnitine-acylcarnitine translocase (CACT) deficiency, and peroxisome biogenesis disorder, Zellweger syndrome. Lignoceric acid (C(24:0)) beta-oxidation in the fibroblasts was stimulated by treatment with BTM, except for Zellweger fibroblasts. In contrasts, palmitic acid (C(16:0)) beta-oxidation was increased (2.8-fold) only in CACT-deficient fibroblasts. In U87 glioblastoma cells, C(24:0) beta-oxidation was also activated by treatment with BTM but C(16:0) beta-oxidation was not. The C(16:0) beta-oxidation was, however, significantly increased in the presence of 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a carnitine palmitoyltransferase I inhibitor. These results indicate that BTM activates peroxisomal but not mitochondrial fatty acid beta-oxidation. In addition, we found that BTM did not upregulate the expression of ABCD2/ALDR, ABCD3/PMP70, ACOX1 and FATP4 genes but slightly increased ACSVL1 gene expression.

Nickel-63 production in copper samples exposed to the Hiroshima atomic bomb: estimation based on an excitation function obtained by neutron irradiation experiments.

The upper and lower limits of the excitation function of the (63)Cu(n,p)(63)Ni reaction were experimentally determined, and the number of (63)Ni nuclei produced in copper samples exposed to atomic bomb neutrons in Hiroshima was estimated by using the experimental excitation functions and the neutron fluences given in the DS02 dosimetry system. The estimated number of (63)Ni nuclei was compared with that measured and with that calculated using the DS02 dosimetry system and the corresponding ENDF/B-VI cross section. In comparison with DS02, there is about a 60% maximum difference in (63)Ni production at the hypocenter when the experimental upper cross section values are used. The difference becomes smaller at greater distances from the hypocenter and decreases, for example, to less than 30 and 5% when using the upper and lower experimental cross sections at 1,000 m, respectively.

Intercomparison study on (152)Eu gamma ray and (36)Cl AMS measurements for development of the new Hiroshima-Nagasaki Atomic Bomb Dosimetry System 2002 (DS02).

In the process of developing a new dosimetry system for atomic bomb survivors in Hiroshima and Nagasaki (DS02), an intercomparison study between (152)Eu and (36)Cl measurements was proposed, to reconcile the discrepancy previously observed in the Hiroshima data between measurements and calculations of thermal neutron activation products. Nine granite samples, exposed to the atomic-bomb radiation in Hiroshima within 1,200 m of the hypocenter, as well as mixed standard solutions containing known amounts of europium and chlorine that were neutron-activated by a (252)Cf source, were used for the intercomparison. Gamma-ray spectrometry for (152)Eu was carried out with ultra low-background Ge detectors at the Ogoya Underground Laboratory, Kanazawa University, while three laboratories participated in the (36)Cl measurement using accelerator mass spectrometry (AMS): The Technical University of Munich, Germany, the Lawrence Livermore National Laboratory, USA and the University of Tsukuba, Japan. Measured values for the mixed standard solutions showed good agreement among the participant laboratories. They also agreed well with activation calculations, using the neutron fluences monitored during the (252)Cf irradiation, and the corresponding activation cross-sections taken from the JENDL-3.3 library. The measured-to-calculated ratios obtained were 1.02 for (152)Eu and 0.91-1.02 for (36)Cl, respectively. Similarly, the results of the granite intercomparison indicated good agreement with the DS02 calculation for these samples. An average measured-to-calculated ratio of 0.98 was obtained for all granite intercomparison measurements. The so-called neutron discrepancy that was previously observed and that which included increasing measured-to-calculated ratios for thermal neutron activation products for increasing distances beyond 1,000 m from the hypocenter was not seen in the results of the intercomparison study. The previously claimed discrepancy could be explained by insufficient understanding of the measured data.

Spatial distribution of soil contamination by 137Cs and 239,240Pu in the village of Dolon near the Semipalatinsk nuclear test site: new information on traces of the radioactive plume from the 29 August 1949 nuclear test.

The village of Dolon located about 60 km northeast from the border of the Semipalatinsk Nuclear Test Site in Kazakhstan is one of the most affected inhabited settlements as a result of nuclear tests by the former USSR. Radioactive contamination in Dolon was mainly caused by the first USSR nuclear test on 29 August 1949. As part of the efforts to reconstruct the radiation dose in Dolon, Cs and Pu in soil samples collected from 26 locations in the vicinity of and within the village were measured to determine the width and position of the center-axis of the radioactive plume that passed over the village from the 29 August 1949 nuclear test. Measured soil inventories of Cs and Pu were plotted as a function of the distance from the supposed center-axis of the plume. A clear shape similar to a Gaussian function was observed in their spatial distributions with each maximum around a center-axis. It was suggested that the plume width that contaminated Dolon was at most 10 km and the real center-axis of the radioactive plume passed 0.7-0.9 km north of the supposed centerline. A peak-like shape with the maximum near the center-axis was also observed in the spatial distribution of the Pu/Cs activity ratio, which may reflect the fractionation effect between Pu and Cs during the deposition process. These results support the recently reported results. The data obtained here will provide useful information on the efforts to estimate radiation dose in Dolon as reliably as possible. Health Phys. 94(4):328-337; 2008.

Atomic bomb induced 152Eu: reconciliation of discrepancy between measurements and calculation.

In order to resolve the discrepancy between the measured and calculated 152Eu activity induced by the atomic bomb at Hiroshima, extremely low background gamma-ray spectrometry was performed for 17 granite samples collected from 134 m to more than 3 km from the hypocenter. Measurements agreed well with theoretical calculations based on DS02 up to 1.4 km from hypocenter.

Atomic force microscopy dissects the hierarchy of genome architectures in eukaryote, prokaryote, and chloroplast.

Because of its applicability to biological specimens (nonconductors), a single-molecule-imaging technique, atomic force microscopy (AFM), has been particularly powerful for visualizing and analyzing complex biological processes. Comparative analyses based on AFM observation revealed that the bacterial nucleoids and human chromatin were constituted by a detergent/salt-resistant 30-40-nm fiber that turned into thicker fibers with beads of 70-80 nm diameter. AFM observations of the 14-kbp plasmid and 110-kbp F plasmid purified from Escherichia coli demonstrated that the 70-80-nm fiber did not contain a eukaryotic nucleosome-like "beads-on-a-string" structure. Chloroplast nucleoid (that lacks bacterial-type nucleoid proteins and eukaryotic histones) also exhibited the 70-80-nm structural units. Interestingly, naked DNA appeared when the nucleoids from E. coli and chloroplast were treated with RNase, whereas only 30-nm chromatin fiber was released from the human nucleus with the same treatment. These observations suggest that the 30-40-nm nucleoid fiber is formed with a help of nucleoid proteins and RNA in E. coli and chroloplast, and that the eukaryotic 30-nm chromatin fiber is formed without RNA. On the other hand, the 70-80-nm beaded structures in both E. coli and human are dependent on RNA.

Urinary bile alcohol profiles in healthy and cholestatic children.

BACKGROUND: Bile alcohols are normal constituents of urine. METHODS: To better understand bile alcohol profile in childhood, urinary specimens from 41 healthy children and 10 children with cholestasis, and 3 healthy adults, were analyzed by GLC and GC-MS. RESULTS: Five bile alcohols, 27-nor-5beta-cholestane-3alpha,7alpha,12alpha,24S,25R-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,24S, 25-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,24S,26-pentol, 5beta-cholestane-3alpha,7alpha, 12alpha,25,26-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,26,27-pentol were identified in all specimens. C(26)-Pentol was the most abundant constituent, constituting 29.5 to 65% of bile alcohols. Among healthy children (n=41), no significant relationship was seen between proportions of the C(26)-pentol and age, but older children (n=15, 6 to 14 years) showed a significantly greater mean percentage of the C(26)-pentol than young children (n=26, 0 to 5 years; 58.1+/-4.23% vs. 46.0+/-9.24%, p<0.001). In children with cholestatic liver diseases, the percentage of C(26)-pentol in urinary bile alcohols was significantly lower than age-matched controls. CONCLUSIONS: There is an increased composition of C(26)-pentol in older children and relatively decreased composition of C(26)-pentol in children with cholestatic liver diseases.

Temperature-sensitive phenotype of Chinese hamster ovary cells defective in PEX5 gene.

SK32 mutant cells, which were isolated as peroxisome-deficient Chinese hamster ovary (CHO) cells by an advantage of a visible peroxisome form of green fluorescent protein (GFP), were found to suffer from a functional loss of PEX5 gene encoding for PTS1R. The sequence analysis of cDNA indicated that PEX5 gene encoded for the two isoforms composed of 603 amino acids (PTS1RS) and 640 amino acids (PTS1RL). The mutation changed glycine to arginine at amino acid position 343 of PTS1RL (corresponding to the position 306 of PTS1RS) in SK32 cells. The mutant cells exhibited a temperature-sensitive (TS) phenotype on the peroxisomal localizations of the recombinant GFP and urate oxidase appending a genuine peroxisome targeting signal 1 (PTS1), a tripeptide of Ser-Lys-Leu (SKL) at the C-terminus, but did not on that of catalase harboring a divergent PTS1, Lys-Ala-Asn-Leu (KANL) sequence. 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase), which harbors an extension sequence (PTS2) at the N-terminus, never appeared to be affected on the peroxisomal localization in the mutant cells. When thiolase was examined on the molecular size in the mutant cells, the enzyme existed as the larger precursor form in the peroxisomes at 37 degrees C and a considerable part (almost half) was converted to the mature size at 30 degrees C. These results indicate that the amino acid substitution, Gly306Arg in PTS1RS and/or Gly343Arg in PTSRL, gives rise to TS phenotype on the peroxisomal translocation of PTS1 proteins and the maturation of PTS2 protein.

Metal-binding properties of phytochelatin-related peptides.

Phytochelatins (PCs, (gamma Glu-Cys)(n)-Gly, n=2-11) are produced by higher plants, algae and some fungi in order to detoxify Cd(2+) by sequestration to form Cd-PCs complexes. In order to investigate what chemical structures of PCs are responsible for their metal-binding ability, various cysteine-rich peptides ((X-Cys)(7)-Gly, X=Glu, Asp, Lys, Gly, Ser and Gln) were chemically synthesized. Water-solubility, metal-binding property, and detoxification effect toward Cd(2+) were analyzed and compared with those of (gamma EC)(7)G. (SC)(7)G and (QC)(7)G were insoluble at pH below 10, and (GC)(7)G was not soluble at any pH between 1 and 12, indicating that charged side chains were at least required for the molecules to be solubilized in aqueous solution. By spectroscopic analyses using DTNB method and UV method, we found that (EC)(7)G and (DC)(7)G had almost equivalent abilities of Cd(2+)-binding as PC ((gamma EC)(7)G), indicating that the distance between each thiol group was not a major factor for the binding to Cd(2+). (beta DC)(7)G and (KC)(7)G interacted to Cd(2+) with fourth coordination as in the case of other soluble PC-related peptides. However, compared to (gamma EC)(7)G, (beta DC)(7)G displayed a slightly weaker binding to Cd(2+), and (KC)(7)G showed a drastic decrease in binding ability. The affinities of PC-related peptides toward Cd(2+) were evaluated as below; (gamma EC)(7)G=(EC)(7)G=(DC)(7)G>(beta DC)(7)G>>(KC)(7)G=weak binding. The results of Cd(2+)-detoxification assays were consistent with the affinity between Cd(2+) and the peptides. We concluded that the structure consisting of thiol and carboxyl groups were essential for the formation of a tight Cd-peptides complex such as Cd-PCs.

Isolation and characterization of psychotrophic bacteria from oil-reservoir water and oil sands.

Four psychrotrophic strains, which grew at 4 degrees C but not at 37 degrees C, were isolated from Japanese oil-reservoir water (strains SIB1, SIC1, SIS1) and Canadian oil sands (strain CAB1). Strains SIB1, SIS1, and CAB1 had a maximum growth rate at 20 degrees C and grew to the highest cell densities at the cultivation temperature of 0-4 degrees C. Strain SIS1 was capable of growing even at -5 degrees C. The growth profile of strain SIC1 was rather similar to that of a mesophilic bacterium. Strains SIB1, SIC1, and SIS1 were identified as members of the genus Shewanella, and strain CAB1 was a member of the genus Arthrobacter. All these strains exhibited weak degradation ability against catechol, a hydroxylated aromatic hydrocarbon, and tributyrin. These strains are expected to be of potential use in the in situ bioremediation technology of hazardous hydrocarbons and esters under low-temperature conditions.

Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A.

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at -5 degrees C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35 degrees C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.

Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart.

Comparison of the conformational stability of an O(6)-methylguanine-DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-MGMT), and its mesophilic counterpart C-terminal Ada protein from Escherichia coli (Ec-AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk-MGMT unfolded at Tm = 98.6 degrees C, which was 54.8 degrees C higher than the unfolding temperature of Ec-AdaC. The maximum free energy (DeltaG(max)) of the proteins were different; the value of Tk-MGMT (42.9 kJ.mol-1 at 29.5 degrees C) was 2.6 times higher than that of Ec-AdaC (16.6 kJ.mol-1 at 7.4 degrees C). The high conformational stability of Tk-MGMT was attributed to a 1.6-fold higher enthalpy value than that of Ec-AdaC. In addition, the DeltaG(max) temperature of Tk-MGMT was considerably higher (by 22.1 degrees C). The apparent heat capacity of denaturation (DeltaC(p)) of Tk-MGMT was 0.7-fold lower than that of Ec-AdaC. These three synergistic effects, increasing DeltaGmax, shifted DeltaG vs. temperature curve, and low DeltaC(p), give Tk-MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk-MGMT possessed higher stability than Ec-AdaC in all conditions studied. These results indicate that the high stability of Tk-MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.