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phoU mutants of bacteria are potentially useful for the removal of inorganic phosphate (Pi) from sewage because they can accumulate a large amounts of polyphosphate (polyP). However, the growth of phoU mutants is severely defective and is easily outgrown by revertant(s) that have lost the ability to accumulate polyP during growth in a nutrient-rich medium. We found that a pseudo-revertant, designated LAP[+], that appeared in a culture of an Escherichia coli phoU mutant that could accumulate polyP even after ten serial passages. Reduction in the expression of the Pi-specific transporter Pst in LAP[+] may contribute to relieving stresses such as excess Pi incorporation that could stimulate reversions. The discovery of a LAP[+] provides a clue to generate phoU mutants that accumulate polyP in a stable manner.

Pentraxin3 (PTX3), a recently discovered inflammatory mediator, is produced abundantly in various cells in atherosclerotic lesions, and therefore, its plasma level could reflect local inflammation at the site of atherosclerotic lesion. The present study evaluated whether plasma PTX3 levels are associated with subclinical atherosclerosis in young subjects with type 1 diabetes.Plasma PTX3 levels, urinary albumin excretion, diabetic retinopathy, and carotid intima-media thickness (IMT) were examined in 78 Japanese type 1 diabetic patients (30 men and 48 women, aged 28.5 ± 5.3 years (±SD), duration of diabetes 19.7 ± 6.5 years).There was statistically significant association between plasma PTX3 levels and Max-IMT (r=0.363, p=0.001). A stepwise multivariate regression analysis including conventional coronary risk factors as independent variables revealed that plasma PTX3 levels (β=0.389, p0.001), duration of diabetes (β=0.256, p=0.035), and serum triglyceride levels (β=0.371, p0.001) were independent determinants of Max-IMT. In addition, plasma PTX3 levels was an independent determinant of urinary albumin excretion, an indicator of diabetic nephropathy (β=0.258, p=0.018). However, there was no significant association between plasma PTX3 levels and diabetic retinopathy.Increased levels of plasma PTX3 are associated with accelerated atherosclerotic change and increased albuminuria in young patients with type 1 diabetes.

DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.

Background The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed. Results A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31. Conclusions In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

We propose the biosensor chip using optical ring resonators. Although the detection of biomarkers for the diagnosis of diseases generally requires high sensitivity of the order of 10-9 g/ml, the detection sensitivity of our device was of the order of 10-7 g/ml. In this paper, we show that 10 or 100 times higher sensitivity than the previous biosensor is accomplished by the following three strategies; (1) using slot-type waveguides, (2) using silicon nitride (SiN) as the waveguide core, (3) improvement of measurement system.

In biological studies, the luminescence from fluorescent proteins or luminescent enzymes is widely used for monitoring a change of environment at a cell. Biomolecules used for this probing, such as Green Fluorescence Protein(GFP) or luciferase molecules can respond to the existence of specific molecules or ions and subsequently emit a photon. The detection of a specific molecule can then be confirmed by detecting the emitted photons efficiently with a photon detector. A highly efficient detection of the luminescence is normally essential to a high sensitivity to the specific molecules or ions. An improvement of the sensitivity can upgrade the capability of detection in a low concentration of sample solution. Therefore, there are many efforts to improve the efficiency of the collection of emitted photons and of the optical coupling to the photon detector (Yotter, 2004).

This paper discusses the features and problems of the Japanese accounting system including related aspects of the tax system, focusing on accounting rules and practices with respect to banks. The Japanese accounting system is based on three closely interrelated legal structures consisting of the Commercial Code, the Securities and Exchange Law, and various tax laws. In recent years, there has been a growing call for review and for establishment of a new framework but which still enables the original aims of each, namely the calculation of profit available for dividends under the Commercial Code, disclosure under the Securities and Exchange Law, and calculation of taxable income under various income tax laws, to be fulfilled. Specific issues concerning the Japanese accounting system include: the introduction of market value accounting (MVA) principles, the amendment of accounting practices to take account of off-balance-sheet transactions, the expansion of the scope of disclosure, review of consolidated accounting standards, improvement of the credit l oss allowance system and the international harmonization of accounting and tax systems. This paper will take up each one of these and examine possible future directions.

Simultaneous pancreas-kidney transplantation is a revolutionary medical procedure to cure diabetes mellitus and its complications in one step. For women with type 1 diabetes mellitus and end-stage renal disease, this procedure not only treats their disease but may also allow them to have children. Worldwide, 77 pregnancies from 43 pancreas-kidney recipients have been reported. Here, we present the first case report of successful pregnancy after simultaneous pancreas-kidney transplantation from a brain-dead donor in Japan. She conceived spontaneously 32 months after transplantation. Her pregnancy course was favorable except for mild rejection of the grafted pancreas and development of gestational diabetes. She delivered a 2882-g healthy infant in October 2010. Pregnancy after simultaneous pancreas-kidney transplantation is discussed.

Intracellular phosphate (P(i) ) is normally maintained at a fairly constant concentration in Escherichia coli, mainly by P(i) transport systems and by the 'phosphate balance' between P(i) and polyphosphate (polyP). We have reported previously that excess uptake of P(i) in a phoU mutant results in elevated levels of polyP. Here, we found that the elevated levels of polyP in the mutant could be reduced by the overproduction of YjbB, whose N-terminal half contains Na(+) /P(i) cotransporter domains. The rate of P(i) export increased when the YjbB overproducer grew on a medium containing glycerol-3-phosphate. These results strongly suggested that YjbB reduced the elevated levels of polyP in the phoU mutant by exporting intracellular excess P(i) .

Fluorescence microscopy (FM) has recently been applied to the detection of airborne asbestos fibers that can cause asbestosis, mesothelioma and lung cancer. In our previous studies, we discovered that the E. coli protein DksA specifically binds to the most commonly used type of asbestos, chrysotile. We also demonstrated that fluorescent-labeled DksA enabled far more specific and sensitive detection of airborne asbestos fibers than conventional phase contrast microscopy (PCM). However, the actual diameter of the thinnest asbestos fibers visualized under the FM platform was unclear, as their dimensions were below the resolution of optical microscopy. Here, we used correlative microscopy (scanning electron microscopy [SEM] in combination with FM) to measure the actual diameters of asbestos fibers visualized under the FM platform with fluorescent-labeled DksA as a probe. Our analysis revealed that FM offers sufficient sensitivity to detect chrysotile fibrils as thin as 30–35 nm. We therefore conclude that as an analytical method, FM has the potential to detect all countable asbestos fibers in air samples, thus approaching the sensitivity of SEM. By visualizing thin asbestos fibers at approximately tenfold lower magnifications, FM enables markedly more rapid counting of fibers than SEM. Thus, fluorescence microscopy represents an advanced analytical tool for asbestos detection and monitoring.

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.

The purified autolytic endo-beta-N-acetylglucosaminidase of Bacillus subtilis AC327 was cleaved with cyanogen bromide, and the N-terminal amino acid sequence of one of the peptide fragments was determined. Then, a DNA fragment containing a part of the glucosaminidase gene was cloned into Escherichia coli JM109 using synthetic oligonucleotides as probes whose sequences had been deduced from the N-terminal amino acid sequence. Zymographic analysis showed that the resultant glucosaminidase-deficient strain lacked a 35-kDa lytic band in addition to a 90-kDa lytic one corresponding to the glucosaminidase. A double mutant strain deficient in the major two autolysins (amidase and glucosaminidase) exhibited greatly impaired motility on a swarm plate whereas the single mutant strains were motile.

A chemotaxis-defective mutant of Enterobacter cloacae IFO3320, designated EC1, was isolated after N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis. Computer-assisted capillary assays showed that EC1 failed to show chemotactic responses to peptone and inorganic phosphate (Pi). Cloning and sequence analysis showed that EC1 is a cheR mutant, suggesting that Pi taxis by E. cloacae is dependent on a methyl-accepting chemotaxis protein(s) (MCP). EC1 was further mutagenized with NTG to construct cheR pstS and cheR pstA double mutants. A recombinant plasmid pECT01.2, which contained the E. cloacae cheR gene, restored the ability of these double mutants to show chemotaxis toward peptone but not Pi. These results suggest that the phosphate-specific transport (Pst) system, together with a MCP(s), is required for detecting Pi in E. cloacae.

We recently reported that the bacterial 50S ribosomal protein L2 binds strongly to silica surfaces even in the presence of high salt concentrations, detergents, and denaturants such as 8 M urea. We designated L2 as Si-tag, a fusion tag for immobilizing functional proteins on silica materials. Here we discuss the remarkable properties of the Si-tag polypeptide in order to understand the mechanism underlying this binding. Experimental and theoretical studies have shown that the 60-aa N-terminal region and the 71-aa C-terminal region, both of which are rich in positively charged residues, lack a well-defined three-dimensional structure under physiological conditions. This lack of a stable tertiary structure suggests that Si-tag belongs to a family of intrinsically disordered (ID) proteins that exist as dynamic ensembles of rapidly fluctuating structures in aqueous solution. Because of its inherent flexibility, Si-tag could form a large intermolecular interface and optimize its structure for surface interactions by conformational adaptation at the binding interface. Such conformational adaptation occurring concomitantly with binding is common to many ID proteins and is called "coupled folding and binding". Through this conformational adaptation, Si-tag could optimize the interactions between its positively charged side chains and ionized surface silanol groups and between its apolar side chains and hydrophobic surface siloxane sites. The cumulative contribution of these contacts would significantly strengthen the binding of Si-tag, resulting in strong, virtually irreversible binding. Our study suggests that flexible ID proteins have tremendous potential for connecting biomolecules to inorganic materials.

We have been demonstrating label-free detection of a variety of antigen–antibody reactions using Si ring optical resonators. Although the detection of biomarkers for the diagnosis of diseases generally requires high sensitivity of the order of 10-9 g/ml, the detection sensitivity of our device is currently of the order of 10-6 g/ml. In this paper, we show that the sensitivity of 10-9 g/ml will be possible by adopting the following four strategies: (1) use of slot-type waveguides with light wavelength of 1.3 µm, (2) improvement of quality factor Q of the ring resonator by smoothing the surface roughness, (3) specific adsorption of the bioreceptor protein to the resonator surface, and (4) maintaining temperature within ±0.005 °C. We have also proposed the on-chip temperature compensation method without the need for temperature control of the sample. By combining the proposed approaches, the sensitivity of the biosensor will be improved by a factor of >100, thus realizing practical application of our Si ring biosensor.

A total synthesis of the natural enantiomer of integerrimine (1), a twelve-membered dilactonic pyrrolizidine alkaloid of retronecine type has been achieved through the enantioselective synthesis and regioselective coupling of (+)-retronecine (4) and (+)-integerrinecic acid (methylthio)methyl ether (6).

Optically active 1,1′-binaphthalene-8,8′-diol is found to bind a variety of amines in CDCl3 or C6D6 solution. Significant chiral recognition (ΔΔG° = ∼1.2 kcal/mol) was observed in the valine derivative 4. A three-point binding motif is assumed.

Necine bases of pyrrolizidine alkaloids, turneforcidine, hastanecine, and platynecine are synthesized from ethyl 1-hydroxy-2,3,5,6-tetrahydro-1H-pyrrolizine-7-carboxylate in racemic form.

Tacrolimus hydrate (FK506), a novel 23-membered macrolide, is an immunosuppressant isolated from Streptomyces tsukubaensis using extensive screening of fermentation products to identify a compound inhibiting the mixed lymphocyte reaction (MLR). The in vitro and in vivo immunosuppressive activities of FK506 were found to be more potent than those of cyclosporine (CyA). The superior immunosuppression with FK506 treatment was also confirmed in the skin allograft model in rats and liver transplantation in dogs. Clinical studies were initiated by Prof. Starzl at the University of Pittsburgh in 1989, and he demonstrated that FK506 surpassed CyA in the incidence of graft survival and the frequency of graft rejection. Multicenter randomized clinical studies, comparing FK506 to CyA corroborated the efficacy of FK506 on the survival of patients and of grafts, and especially on the appearance of severe refractory graft rejection. FK506 was marketed in 1993 in Japan, and was followed in 1994 in the U.S.A., U.K. and Germany. The mechanism of action of this compound was clarified by the endeavors of Prof. Schreiber, who demonstrated the existence of a binding protein for FK506 called FKBP, similar to cyclophilin for CyA. The FK506/FKBP complex binds with calcineurin, a serine/threonine phosphatase to inhibit the translocation of NFAT into the nucleus, resulting in inhibition of transcription of IL-2 mRNA. FK506 displays potent immunosuppressant activity, and contributes not only to the progress of transplantation therapy for clinical studies, but also to the clarification of signal transduction in T cell activation for basic science.

A monodentate phosphine, 8-diphenylphosphino-8'-methoxy-1, 1'-binaphthyl, was shown to be active as a ligand in palladium-catalyzed reactions, including the reduction of allylic carbonates with formic acid, hydrosilylation of a terminal olefin, and hydroboration of but-1-en-3-yne, even though it has a highly hindered lone pair.

A down-converter insensitive to 2nd and 3rd harmonics and a digital assisting circuit that cancels residual sensitivity due to device mismatches are proposed. Measured 2nd and 3rd harmonic rejection ratios of over 60 dB are achieved. This performance allows a simple RF filter, and it is effective to reduce the die size.

The structures of mechanically alloyed (MA) and melt-spun (MS) NixZr1-x (x = 0.28, 0.33, 0.40) amorphous alloys were investigated by X-ray diffraction. The interatomic distances, rNiZr and rZrZr, were calculated from the deconvolution of the principal peak of the radial distribution function (RDF) using Gaussian functions. The distances were nearly equal for the amorphous alloys of the same composition prepared by the two methods, i.e., 2.69 and 2.67 A and 3.28 and 3.22 A on average for the MA and MS specimens, respectively.

A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.

A total synthesis of the natural enantiomer of integerrimine (1), a twelve-membered dilactonic pyrrolizidine alkaloid of retronecine type has been achieved through the enantioselective synthesis and regioselective coupling of (+)-retronecine (4) and (+)-integerrinecic acid (methylthio)methyl ether (6).

The phosphate (P(i))-binding protein PstS is a member of a family of periplasmic proteins that act as high-affinity receptors for active transport systems in bacteria. PstS protein purified from Pseudomonas aeruginosa was immobilized to N-hydroxysuccinimide-activated Sepharose, packed into a plastic column (5 x 70 mm), and examined for its potential ability to remove P(i) from water. The PstS-Sepharose column completely removed P(i) from 32P-labeled pond water containing about 0.5 microM P(i) (0.015 mg P per liter). More than 90% of 32P-P(i) that was retained in the column could be eluted by washing with low-pH water.

Fluorescence microscopy (FM) is one of the most important analytical tools in modern life sciences, sufficiently sensitive to allow observation of single molecules. Here we describe the first application of the FM technique for the detection of inorganic environmental pollutants—airborne asbestos fibers that can cause asbestosis, mesothelioma, and lung cancer. In order to assess FM capabilities for detecting and counting asbestos fibers, we screened E. coli lysate for proteins that bind to amphibole asbestos. In combination with the previously discovered E. coli protein DksA (Kuroda et al., Biotechnol. Bioeng. 2008, 99, 285−289) that can specifically bind to chrysotile, the newly identified GatZ protein was used for selective and highly sensitive detection of two different asbestos types. Our novel FM-based method overcomes a number of limitations of the commonly used phase-contrast microscopy (PCM) method, offering a convenient alternative to PCM for airborne asbestos monitoring.

The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. However, these conventional methods have problems with regard to their measurement time or sensitivity. We recently obtained a mutant firefly luciferase that has a luminescence intensity over 10-fold higher than that of the wild type. Therefore, we developed a new endotoxin detection method that combines the Limulus reaction and bioluminescence using mutant luciferase. The new method detects 0.0005 EU/ml of endotoxin within 15 min.

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. Methodology/Principal Findings Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. Conclusions/Significance Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B. subtilis cw1A gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL. In contrast to the molecular mass of 29 919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein. Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-l-alanine amidase. A 32-kDa precursor protein was detected on zymography of a crude cell homogenate. Some of the enzymatic properties of CWLA are also described.

The precipitation behavior of 1,1-di(p-hydroxyphenyl)cyclohexane (DHC) from acetone solutions containing d-Limonene (1-methyl-4(I-methylethenyl)cyclohexene) was studied. From the pure acetone solution or the solutions containing a small amount of d-Limonene crystals (B) precipitated, which clathrate only acetone with a guest/host (G/H) molar ratio of 1.0. However, when thed-Limonene concentration is increased to more than ca. 2 mol/L, crystals (A) precipitated which had a different habit from the B crystals. In the A crystalsd-Limonene is clathrated together with a large amount of acetone and the G/H value ofd-Limonene increases with the concentration in the solution up to the maximum value of 0.2. As the diffraction patterns of the A and B crystals are similar, it is assumed that a part of the acetone molecules in the B crystals are replaced byd-Limonene molecules. The acetone in the A crystals escapes rapidly, but thed-Limonene remains for a long time. This may indicate that the large molecule ofd-Limonene cannot diffuse rapidly within the host lattice owing to three-dimensional hindrance. It was clear that the solubility of the A crystals is higher than that of the B crystals and the transformation from the'metastable A to the stable B crystals proceeds during the crystallization of A crystals.

BACKGROUND The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. METHODS Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. RESULTS p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134+/-297 vs. 1477+/-145 IEQ/g or 65,012+/-9385 vs. 45,700+/-5103 IEQ/pancreas; P

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

We examined performance of mice in discrimination of liquor odors by Y-maze behavioral assays. Thirsty mice were initially trained to choose the odor of a red wine in the Y-maze. After successful training (>70% concordance for each trained mouse), the individual mice were able to discriminate the learned red wine from other liquors, including white wine, rose wine, sake, and plum liqueur. However, when the mice were tested to distinguish fine differences between 2 brands of red wine, their performance significantly varied among the individual trained mice. Among 10 mice tested, 2 mice were able to discriminate between the red wines (>75% concordance) whereas 6 mice failed to distinguish between them (50-67% concordance, where chance could be assumed to be 50%). More importantly, 2 other mice exhibited lower than 30% concordance, indicating that they were more attracted to the nonrewarded red wine compared with the learned one. This result suggested that the individual mice directed attention to different subsets of volatile components emanating from the rewarded red wine, when they were trained to choose the liquor odor in the Y-maze. Selective attention of mice was also observed in Y-maze behavioral assays using the mixtures of 3 or less pure odorants. Additionally, we also observed that the olfactory attention of mice could be modified through their learning experiences.

SUMMARY: We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241--248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed δ promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.

A DNA fragment containing the flaD locus of Bacillus subtilis, which had been cloned into plasmid pAC3, was subcloned into an M13 phage and sequenced. The sequence contained five open reading frames (ORFs), of which ORF2 was the flaD gene. Unexpectedly, the sequence of the flaD locus was identical to that of sin [sporulation inhibition gene; Gaur, N. K., Dubnau, E.Smith, I. (1986). Journal of Bacteriology 168, 860-869]. A B. licheniformis homologue (flaL) of the B. subtilis flaD locus was cloned into pUC19 and identified by colony hybridization. The B. licheniformis DNA was subcloned and sequenced. Two ORFs (ORF1, or L-ORF1; and ORF2, or flaL) were detected, encoding 58 and 111 amino acid residues, respectively. These are almost identical in length to ORF1 (D-ORF1; 57 amino acids) and flaD (111 amino acids) on the fragment of B. subtilis DNA. The overall interspecies differences between the nucleotide sequences of D-ORF1 and L-ORF1, and those of flaD and flaL, were 42% and 11%, respectively, and the differences in the predicted amino acid sequences were 50% and 7%, respectively. The regions 3' of the ORFs (flaL and flaD) in both species resemble rho-independent terminators of transcription. The characteristics of the amino acid sequences are also discussed.

Fatigue damage of interfacial delamination of notched CFRP has been observed by a scanning acoustic microscope (SAM), and the delamination resistances against fatigue cycles are discussed.By using a pulse wave of 50MHz, the delamination images at each interfacial site were clearly observed by a SAM. The delamination was quite different at each interface, and the interfacial delamination ahead of a notch was hardly observed between the 1st and the 2nd laminae but firstly observed between the 2nd and the 3rd laminae. The length of interfacial delamination evaluated in terms of specimen compliance was well corresponded with the saw-cut notch of the equal length, and the delaminated portion lost its load carrying capacity. Axial splits in 0° plies had the effect of separating the 0° plies into two outer strips containing the notch and an inner strip without a notch, thereby reducing the notch-tip stress concentration. The growth rate of interfacial delamination judged in terms of the compliance change was accelerated with an increase in notch length as well as with an increase in maximum ligament stress. The notch sensitivity of CFRP was quite different from the one of uniform materials.

In the current studies, we found that the DksA protein from Escherichia coli binds strongly to chrysotile, which is the most commonly used form of asbestos. We developed a convenient colorimetric assay for chrysotile using a fusion of DksA and alkaline phosphatase along with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium as substrates. Also, using a fusion of DksA and green-fluorescent protein, we were able to detect chrysotile by fluorescence microscopy.

A microchannel reactor system was used in a biodesulfurization process in which the rate of biodesulfurization in the oil/water phase of the microchannel reaction was more than nine-fold that in a batch (control) reaction. In addition, the microchannel reaction system using a bacterial cell suspension degraded alkylated dibenzothiophene that was not degraded by the batch reaction system. This work provides a foundation for the application of a microchannel reactor system consisting of biological catalysts using an oil/water phase reaction.

A rapid method for detection of Escherichia coli O157: H7 using multiplex PCR was developed. Two oligonucleotide primer pairs were used for simultaneously detection of vt encoding verotoxin genes for virulence factor and rfb(O157) encoding the O-antigen specific for E. coli O157: H7. Multiplex PCR generated two products of 215 bp and 420 bp for vt and rfb(O157), respectively. Multiplex PCR detected reference strain O157: H7 (NF-7777) with a sensitivity of 10(5) CFU per ml with no amplification of other 15 pathogenic bacteria. After incubation of 10(2) CFU/25 gram raw meat in tryptic soy broth at 37 degrees C for 8 hours, multiplex PCR conducted with the addition of 100 mg bovine serum albumin produced the two specific PCR products for E. coli O157: H7. This modified multiplex PCR is a rapid, sensitive, and specific technique for detecting and differentiating E. coli O157: H7 and has the potential to be used as an alternative to conventional methods for the screening of O157: H7 strains isolated from raw meat.

Firefly luciferase is widely used for enzymatic measurement of ATP, and its gene is used as a reporter for gene expression experiments. From our mutant library, we selected novel mutations in Photinus pyralis luciferase with higher luminescence intensity. These included mutations at Ile423, Asp436, and Leu530. Luciferase is structurally composed of a large N-terminal active site domain (residues 1-436), a flexible linker (residues 436-440) peptide, and a small C-terminal domain (residues 440-550) facing the N domain. Thus, the mutations are located at the junction of the N-terminal domain and the flexible linker, in the flexible linker peptide, and in the tip of the C-terminal domain, respectively. Substitution of Asp436 with a nonbulky amino acid such as Gly remarkably increased the substrate affinity for ATP and d-luciferin. Substitution of Ile423 with a hydrophobic amino acid such as Leu and that of Leu530 with a positively charged amino acid such as Arg increased the substrate affinity and the turnover rate. Combining these mutations, we obtained luciferases that generate more than 10-fold higher luminescence intensity than the wild-type enzyme.