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Pulmonary surfactant proteins (SP) A and D play important roles in the innate immune system of the lung. These proteins belong to the collectin subgroup in which lectin domains are associated with collagenous structures. To obtain a better understanding of how lung collectins modulate cellular responses, the authors investigated whether SP-A interacts with the toll-like receptor 2 (TLR2). SP-A bound to TLR2 and inhibited interactions between TLR2 and TLR2-ligands such as peptidoglycan (PGN) and zymosan. NF-κB activation and tumour necrosis factor-α expression induced by PGN or zymosan were significantly inhibited in the presence of SP-A. Lung collectins may act as inhibitors of lung inflammation in respiratory infections. The authors also examined the effects of lung collectins on the phagocytosis of bacteria by alveolar macrophages. Lung collectins enhanced the uptake of S. pneumoniae or M. avium by alveolar macrophages. It was demonstrated that the direct interaction of lung collectins with macrophages resulted in increased cell surface expression of scavenger receptor A or mannose receptor, which are responsible for phagocytosis. This study has emphasized the biological relevance of SP-A and SP-D against various respiratory infections, however, a more complete understanding of the molecular mechanism is required.

In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31–5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method ( R 2 =0.973).

Toll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-kappaB activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-kappaB activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN.

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.

Lipopolysaccharide (LPS) has been known to induce inflammation by interacting with CD14, which serves as a receptor for LPS. Mannose-binding protein (MBP) belongs to the collectin subgroup of the C-type lectin superfamily, along with surfactant proteins SP-A and SP-D. We have recently demonstrated that SP-A modulates LPS-induced cellular responses by interaction with CD14 (H. Sano, H. Sohma, T. Muta, S. Nomura, D. R. Voelker, and Y. Kuroki, J. Immunol. 163:387–395, 2000) and that SP-D also interacts with CD14 (H. Sano, H. Chiba, D. Iwaki, H. Sohma, D. R. Voelker, and Y. Kuroki, J. Biol. Chem. 275:22442–22451, 2000). In this study, we examined whether MBP, a collectin highly homologous to SP-A and SP-D, could bind CD14. Recombinant rat MBP-A bound recombinant human soluble CD14 in a concentration-dependent manner. Its binding was not inhibited in the presence of excess mannose or EDTA. MBP-A bound deglycosylated CD14 treated withN-glycosidase F, neuraminidase, andO-glycosidase, indicating that MBP-A interacts with the peptide portion of CD14. Since LPS was also a ligand for the collectins, we compared the characteristics of binding of MBP-A to LPS with those of binding to CD14. MBP-A bound to lipid A fromSalmonella entericaserovar Minnesota and rough LPS (S. entericaserovar Minnesota Re595 andEscherichia coliJ5, Rc), but not to smooth LPS (E. coliO26:B6 and O111:B4). Unlike CD14 binding, EDTA and excess mannose attenuated the binding of MBP-A to rough LPS. From these results, we conclude that CD14 is a novel ligand for MBP-A and that MBP-A utilizes a different mechanism for CD14 recognition from that for LPS.

A novel bioluminescence (BL) assay of pyruvate phosphate dikinase (PPDK) has been developed using the firefly luciferin–luciferase reaction. PPDK catalyzes the formation of ATP from AMP, PPi (diphosphate) and phosphoenolpyruvate (PEP). The BL assay for PPDK was highly sensitive with a low background because it was not affected by adenylate kinase, which generates ATP from 2 mol of ADP and is present in various microorganisms. The proposed assay was performed as follows: 10 μl of standard PPDK solution was added to a microtiter plate, followed by addition of 100 μl of bioluminescent solution (containing AMP, PPi, PEP, Mg 2+ , luciferin and luciferase). The BL intensity was integrated for 5 s after incubation for 15 min. The range of log–log calibration linearity and 3S.D. detection limit of PPDK ranged from 3.5×10 −20 to 4.5×10 −16 and 1.36×10 −20 mol per assay, respectively. The within-assay precision for PPDK was 1.2–2.8% ( n =8). The BL intensity was stable for at least 120 min at 37°C. The proposed assay to was applied to a BL enzyme immunoassay (EIA) for α-fetoprotein (AFP) and insulin using PPDK as the enzyme label. The detection limits for AFP and insulin were 2.79×10 −18 and 9.26×10 −17 mol per assay, respectively. Serum samples could accurately be analyzed by the BL-EIA. A high degree of correlation was observed between the values obtained using the proposed BL-EIA and conventional methods.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening, interstitial lung disease of unknown etiology. For optimal therapeutic management of IPF an accurate tool is required for discrimination between reversible and irreversible types of the disease. However, such noninvasive tools are few, and even with high-resolution computed tomography (HRCT), which is the most trusted method for doing so, the nature of the disease activity in IPF cannot always be accurately predicted. The aims of the present study were to assess the values of surfactant protein (SP)-A and SP-D in semiquantifying the extent of disease in IPF and in predicting deterioration in restrictive pulmonary function and survival over a follow-up period of 3-yr. SP-A and SP-D in sera were measured with enzyme-linked immunosorbent assays as previously described. Fifty-two IPF patients were studied to evaluate the association between serum SP-A and SP-D and disease extent on HRCT, deterioration in pulmonary function, and survival during 3 yr of follow-up. Both SP-A and SP-D concentrations were significantly correlated with the extent of alveolitis (a reversible change), whereas they did not correlate with the progression of fibrosis (an irreversible change). The SP-D concentration, unlike that of SP-A, was also related to the extent of parenchymal collapse and the rate of deterioration per year in pulmonary function. The concentrations of SP-A and SP-D in patients who died within 3 yr were significantly higher than in patients who were still alive after 3 yr. We propose that assays of SP-A and SP-D in sera from IPF patients are useful tools for understanding some pathologic characteristics of the disease, that SP-D may be a good predictive indicator of the rate of decline in pulmonary function, and that a combination of the assays for SP-A and SP-D may be helpful in predicting the outcome of patients with IPF.

Various thermophilic bacteria were analyzed by Southern hybridization analysis using oligonucleotide probes coding for the pyruvate phosphate dikinase (PPDK) gene from Clostridium symbiosum, and positive hybridization signals were observed in the chromosomal DNAs from Microbispora rosea subsp. aerata (IFO 14047). PPDK activity was detected in lactose induced cells and the enzyme was purified to homogeneity. The molecular mass of PPDK was estimated to be 230000 by gel filtration chromatography and 91000 by SDS-PAGE, suggesting that PPDK is a dimeric enzyme. This enzyme was specific for adenine nucleotide and the apparent Km values for AMP, PPi, and phosphoenolpyruvate were 5, 38, and 280 microM, respectively. It was stable in the pH range 6 to 11, and retained 80% activity after 60 min heat treatment at 60 degrees C. We cloned the PPDK gene from M. rosea. It consists of 878 amino acids with a molecular mass of 95514. Sequence comparison indicates around 50% similarity with other PPDKs and it has all the highly conserved regions of the related enzymes. We expressed the PPDK gene in Escherichia coli and obtained enzymatically active protein.

Pituitary adenylate cyclase activating polypeptide 38 (PACAP38) is a novel peptide hormone and has a variety of biological actions. In studies of the physiological behavior of endogenous PACAP, the determination of PACAP38 levels in biological materials requires a highly sensitive and specific method. Therefore, we developed a sensitive bioluminescent enzyme immunoassay (BL-EIA) for PACAP38 using acetate kinase (AK) as a reporter enzyme and the luciferin–luciferase reaction. Accordingly, we developed BL-EIA using biotinylated PACAP38 as a labeled antigen and biotinylated AK–streptavidin complex as a detector of biotin on solid phase. The measurable range was 62–16000 pg ml −1 for PACAP38 by the proposed BL-EIA. The PACAP38 concentration of various tissue extracts of rat and plasma of rat and human could be measured by the proposed BL-EIA. Furthermore, a high degree of correlation for various samples was found between the results obtained by BL-EIA and radioimmunoassay.

A bioluminescent assay of acetate kinase (AK) have been developed using thermostable mutant firefly luciferase. The principle of the method is as follows: AK catalyzes the formation of ATP from acetylphosphate and ADP, which is readily consumed by the luciferin–luciferase reaction resulting in bioluminescence. AK activity was determined by measuring the light generated by the luciferase reaction. In this method, we have been able to detect 8.5×10−21 mol of AK. The bioluminescence was stable for at least 100 min at 37°C. This bioluminometric assay of AK was applied to the enzyme immunoassay of human growth hormone (hGH) and human chorionic gonadotropin (hCG), using biotinylated AK and streptavidin. In these enzyme immunoassays, the measurable ranges of hGH and hCG were 78–20,000 pg ml−1 and 39–20,000 μIU ml−1, respectively. This assay system can be applied to various enzyme immunoassays with high sensitivity.

A novel and effective treatment of biological samples with a combination of adenosine phosphate deaminase and apyrase was developed for reducing extracellular ATP, which has been a major problem encountered in improving the sensitivity of assays for intracellular ATP by the firefly luciferin-luciferase (L-L) method. Under the enzymatic reaction conditions, ATP and the related adenosine derivatives were converted to IMP, which are not active to the L-L system. In the model system (3.2 x 10(-8) M ATP in 1% yeast extract solution) the treatment with adenosine phosphate deaminase resulted in the reduction of ATP to 1.3 x 10(-11) M, and the concomitant use of apyrase lowered the concentration to 3.3 x 10(-13) M. The treatment (0.05 U/ml of adenosine phosphate deaminase and apyrase) was applied to the detection of bacteria in broth by the L-L method, affording the detection of 42 colony-forming unit (CFU)/ml of Escherichia coli and 10 CFU/ml of Staphylococcus aureus in the broth.

Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

Streptavidin-biotin system has been introduced to a bioluminescent enzyme immunoassay using biotinylated acetate kinase and recombinant firefly luciferase. The streptavidin-biotin based bioluminescent enzyme immunoassays (BLEIAs) for thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG) were developed in which the complexes of biotinylated acetate kinase, streptavidin, and biotinylated antibody were used. In BLEIA for TSH, acetate kinase activity was determined by endpoint assay and for hCG the enzyme activity was measured by rate assay. The measurable ranges of TSH and hCG were 0.02 μIU/ml to 50 μIU/ml and 0.003 to 2 μIU/ml, respectively. The proposed streptavidin-biotinylated acetate kinase system was shown to be applicable to several sandwich type enzyme immunoassays employing biotinylated antibody with high sensitivity.

A sensitive bioluminescent enzyme immunoassay (BLEIA) has been developed using acetate kinase(AK) as a new label enzyme. The bioluminescent assay of AK was done by firefly luciferase for measuring ATP produced by enzymatic reaction of AK using acetylphosphate and ADP as substrate. The detection limit of AK was 60 zmol/assay(1.2 amol/ml) and the coefficient of variation (CV, n=5) for each point, ranged from 2.1 to 8.3%. The proposed bioluminescent assay of AK was very simple, rapid and highly sensitive. The AK assay developed here, was applied to BLEIA of 17 α-hydroxyprogesterone, rabbit IgG and human thyroid stimulating hormone(TSH) as model compound for competitive and immunometric immunoassay.

タンパク質工学的手法により作製された各種変異型ホタルルシフェラーゼを精製し,その酵素化学的性質を調べた.至適pH及び基質特異性は各変異体とも野生型ルシフェラーゼと同じであった.一方で耐熱性及びpH安定性は野生型ルシフェラーゼに比べ優れていた.特にAla217Leu変異型ヘイケホタルルシフェラーゼは最も安定であり,50℃,20分間の熱処理後も50%の活性が残存していた.大腸菌による大量生産が可能なThr217Ile変異型ゲンジホタルルシフェラーゼを,アデノシン三リン酸(ATP)測定及びアセテートキナーゼ(AK)の活性測定に応用した.生物発光法によるATPの定量域は2.0×10-14～2.0×10-10Mであった.AKの活性測定は酵素反応によるATP産生とルシフェリンールシフェラーゼ系を共役させることにより測定した.AKの検出限界は8.6×10-21mol/assay(5200分子)と高感度なAK活性測定法を確立することができた.

This paper describes our believable judge bot ICE-CIG2011 that has an ability to learn tactics from a judge player and an ability to judge an opponent character as a human or a bot. We conjecture that a bot with these two abilities should be considered human-like in a competition environment, such as BotPrize, where human players participate to compete not only for being the most human-like player but also the best judge. Main contributions of this work lie in our mechanisms for achieving these two abilities. To achieve the former ability, we develop a system and GUI that allow a selected judge player — whose role is to train ICE-CIG2011 — to control his or her character by only deciding which tactic to use under a given situation. We then obtain the judge's tactic log and use it for training tactic selection of ICE-CIG2011 with neuro evolution of augmenting topologies. To achieve the latter ability, we acquire additional logs when the judge character interacts with other opponent characters. In order to represent the play of a known (bot or human) character, we train a neural gas — a kind of self-organizing neural network — from its log. For an unknown character, once its neural gas is trained after a certain period of observation, ICE-CIG2011 decides if it is a human or bot by using the if-nearest-neighbor algorithm; this algorithm considers the majority in the labels of the if-nearest neural gases, of known characters, to the neural gas of that unknown character. Experimental results are given and discussed concerning these two abilities of ICE-CIG2011.

The genomic DNA for the alkaline protease (Alp) of the fungus Aspergillus oryzae was isolated using synthetic oligonucleotides as hydridization probes, and the complete nucleotide sequence was identified. The Alp gene is 1374 nucleotides long and contains three introns, one of which is in the pro region and two in the mature coding region. Sequences related to the TATA box (TATAAAT) and the CAAT box (CCAAAT) were found in the 5'-noncoding region. Primer extension analysis showed that three transcriptional start points are present.

The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.

A 22-year-old man was admitted to our hospital because of high fever and shortness of breath. A chest CT showed centrilobular nodules and ground-glass attenuation in both lungs. Transbronchial lung biopsy specimens showed alveolitis including the infiltration of mononuclear cells and granulomas. He responded to treatment, but upon returning home the radiological findings and clinical symptoms reappeared. We focused on a dehumidifier because it had been used continuously in his room. Thermoactinomyces vulgaris presented in the air filter of the dehumidifier. The test of precipitation in response to Thermoactinomyces vulgaris was positive. The condition inside the dehumidifier of a high temperature and high humidity were suitable for the proliferation of Thermoactinomyces vulgaris. We diagnosed hypersensitity pneumonitis due to a dehumidifier contaminated by Thermoactinomyces vulgaris.

In this paper we present a new method for finding frequent patterns from tree-structured data, where a frequent pattern means a subgraph which frequently occurs in a given tree-structured data. We make use of a data compression method called TGCA for tree-structured data. Improving manipulation of large scaled data by compressing them has been investigated in previous studies, such as keyword search in plain texts, and frequent itemset mining from transaction data, but it has not been applied to finding frequent patterns from tree-structured data in the best of our knowledge. The TGCA algorithm is obtained by modifying the SEQUITUR algorithm for plain texts so that it can compress tree-structured data, and we show that we can count occurrences of patterns in the original data by using the data compressed by TGCA without expanding it. This is the reason why our method improves the efficiency of finding frequent patterns. The advantage of our method is shown in some experiments in the case that the data can be compressed in some good compression ratios.

To produce Aspergillus oryzae alkaline protease (Alp) in an osmophilic yeast Zygosaccharomyces rouxii, we constructed an expression plasmid consisting of the Z. rouxii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the prepro-Alp cDNA of A. oryzae, the whole sequence of Z. rouxii plasmid pSR1, and the G418 resistant gene. The resulting plasmid, when introduced into Z. rouxii cells, directed the secretion of a large amount (about 300 mg/l) of Alp into the culture medium. The N-terminus and specific activity of the enzyme were identical to those of A. oryzae Alp.

We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 x 10(-20) and 2.05 x 10(-20) mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.

To improve the practical usefulness of the firefly luciferase, we performed gene chimerization between Photinus pyralis luciferase and a thermostable variant of Luciola cruciata luciferase. One chimeric luciferase showed low K(m) value for substrate ATP and similar stability to thermostable L. cruciata luciferase. We then introduced random mutations in the corresponding gene and screened for increased catalytic efficiency. Amino acid replacement of Thr219, Val239 and Val290 affected the kinetic parameters. Therefore, we combined these three mutations. One mutant, ABcT219I,V239I, showed high catalytic efficiency comparable to P. pyralis luciferase and high stability similar to thermostable L. cruciata luciferase. The pH-dependence of the bioluminescence emission spectra was also examined. In contrast to wild-type firefly luciferases characterized to date, the mutant did not show the pH-dependent red spectrum shift.

Pulmonary surfactant protein A (SP-A) plays an important role in modulation of the innate immune system of the lung. Peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, is known to elicit excessive proinflammatory cytokine production from immune cells. In this study we investigated whether SP-A interacts with PGN and alters PGN-elicited cellular responses. Binding studies demonstrate that PGN is not a ligand for SP-A. However, SP-A significantly reduced PGN-elicited tumor necrosis factor alpha (TNF-alpha) secretion by U937 cells and rat alveolar macrophages. The inhibitory effect on TNF-alpha secretion was dependent upon SP-A concentrations in physiological range. Coincubation of SP-A and PGN with human embryonic kidney 293 cells that had been transiently transfected with the cDNA of Toll-like receptor 2 (TLR2), a cell signaling receptor for PGN, significantly attenuated PGN-induced nuclear factor-kappaB activity. SP-A directly bound to a soluble form of the recombinant extracellular TLR2 domain (sTLR2). Coincubation of sTLR2 with SP-A significantly reduced the binding of sTLR2 to PGN. These results indicate that the direct interaction of SP-A with TLR2 alters PGN-induced cell signaling. We propose that SP-A modulates inflammatory responses against the bacterial components by interactions with pattern-recognition receptors.

Toll-like receptor 2 (TLR2) and CD14 function as pattern recognition receptors for bacterial peptidoglycan (PGN). TLRs and CD14 possess repeats of the leucine-rich motif. To address the role of the extracellular domain of TLR2 in PGN signaling, we constructed CD14/TLR2 chimeras, in which residues 1-356 or 1-323 of CD14 were substituted for the extracellular domain of TLR2, and five deletion mutants of TLR2, in which the progressively longer regions of extracellular TLR2 regions were deleted. PGN induced NF-kappaB activation in HEK293 cells expressing TLR2 but not in cells expressing CD14/TLR2 chimeras. The cells transfected with a deletion mutant TLR2(DeltaCys30-Ile64) as well as TLR2(DeltaCys30-Asp160) and TLR2(DeltaCys30-Asp305) failed to respond to PGN, indicating the importance of the TLR2 region Cys(30)-Ile(64). Although TLR2(DeltaCys30-Ser39) conferred cell responsiveness to PGN, the cells expressing TLR2(DeltaSer40-Ile64) failed to induce NF-kappaB activation. In addition, NF-kappaB activity elicited by PGN was significantly attenuated in the presence of synthetic peptide corresponding to the TLR2 region Ser(40)-Ile(64). From these results, we conclude that; 1) CD14 cannot functionally replace the extracellular domain of TLR2 in PGN signaling; 2) the TLR2 region Cys(30)-Ser(39) is not required for PGN recognition; 3) the TLR2 region containing Ser(40)-Ile(64) is critical for PGN recognition.

The production mechanism of shoyuflavones, conjugated ethers of isoflavones with tartaric acid and isolated from fermented soy sauce, was studied. In the high molecular weight fraction of the culture extract of Aspergillus oryzae, genistein was transformed into shoyuflavone B in the presence of (±)-trans-epoxysuccinic acid but not in the low molecular one. Asp. sojae and Asp. tamarii showed high activity similar to Asp. oryzae but none of Asp. niger, Rhizopus oligosporus, and Mucor praini did. The contents of epoxysuccinic acids in the starting materials of soy sauce and the cultures of various Asp. fungi were determined as dimethyl 2-chloro-3-hydroxysuccinate derivatives by GC-MS. Although epoxysuccinic acids were contained in Asp. oryzae, Asp. sojae, and Asp. tamarii cultures, they were not found in soybeans and wheat. A possible producing mechanism for shoyuflavones by enzymatically conjugating isoflavones to (±)-trans-epoxysuccinic acid with ether linkage was suggested. Keywords: Isoflavone; trans-epo...

Surfactant proteins A and D (SP-A and SP-D) are structurally related members of the collectin family found in the alveolar compartment of the lung. SP-A binds dipalmitoylphosphatidylcholine (DPPC) and galactosylceramide (GalCer), induces liposome aggregation, and regulates the uptake and secretion of surfactant lipids by alveolar type II cells in vitro. SP-D binds phosphatidylinositol (PI) and glucosylceramide. The purpose of this study was to identify a critical stretch of primary sequence in the SP-A region Cys(204)-Phe(228) and the SP-D region Cys(331)-Phe(355) that is involved in protein-specific lipid and type II cell interactions. Chimeras ad1 and ad2 were constructed with rat SP-A/SP-D splice junctions at Cys(218)/Gly(346) and Lys(203)/Cys(331), respectively. Chimera ad1 but not ad2 retained DPPC liposome binding activity. Both chimeras retained significant binding to GalCer liposomes. Chimera ad1 did not bind to PI, whereas chimera ad2 acquired a significant PI binding. Both chimeras failed to induce liposome aggregation and to interact with alveolar type II cells. In addition, monoclonal antibody 1D6 that blocks specific SP-A functions did not recognize either chimera. From these results, we conclude that (1) the SP-A region Leu(219)-Phe(228) is required for liposome aggregation and interaction with alveolar type II cells, (2) the SP-A region Cys(204)-Cys(218) is required for DPPC binding, (3) the SP-D region Cys(331)-Phe(355) is essential for minimal PI binding, and (4) the epitope for mAb 1D6 is located at the region contiguous to the SP-A region Leu(219)-Phe(228).

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.

The pyruvate kinase of Microbispora thermodiastatica was purified to homogeneity and some properties of the enzyme were characterized. The molecular weight of the enzyme by gel filtration is 277,000. The subunit molecular weight is 55,000 by SDS-polyacrylamide gel electrophoresis, and only one N-terminal amino acid sequence was obtained. It had a pH optimum around pH 4.5 to 7.0 and was stable over the range of pH 4.0-8.0. The enzyme is thermostable and no activity was lost after heat treatment at 55 degrees C for 60 min. AMP activated this enzyme and the saturation curve of the enzyme for PEP changed from sigmoidal type to hyperbolic type in the presence of AMP.

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.

(1992). Enhancement of the Thermostability of the Alkaline Protease from Aspergillus oryzae by Introduction of a Disulfide Bond. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 2, pp. 326-327.

Bromoperoxidases were found in coralline algae (Corallinaceae, Rhodophyta) collected from seasides in Japan, and high enzyme activities were observed in Corallina officinalis, Corallina pilulifera and Amphiroa zonata. The optimum pHs of the enzymes from coralline algae were around 6.0. The enzymes were specific for I− and Br−, and did not act on Cl− and F−. The enzymes purified from Corallina pilulifera and Amphiroa ephedraea catalyzed the brominations of phenol and O-hydroxybenzyl alcohol in the presence of Br− and H202 to form the same product, 2,4,6-tribromophenol.

1. Die hintere Wurzel des Halsnerven des Hundes enthalt eine verschiedene Menge von spinalparasympathischen Fasern; die Menge derselben ist je nach den Individuen stark variabel. 2. Die Zwischenschaltganglienzelle fur den Spinalparasympathicus im Spinalganglion wurde mittels der Methode der retrograden Veranderung nach der Durchtrennung des Fortsatzes bestimmt. 3. Die grosen und mittelgrosen Zellen und ein Teil der kleinen Zellen zeigten nach der Durchtrennung der hinteren Wurzel deutliche, aber eigentumliche retrograde Veranderung, die schon am 4. Tage nach der Operation deutlich wird. Ein Teil der kleinen Zellen blieb ganz unverandert. 4. Alle Zellen, abgesehen von den kleinsten Zellen, zeigen nach der Durchtrennung des peripheren Nerven retrograde Veranderung, die aber erst nach 1 Woche deutlich wird; dagegen zeigt ein Teil der kleinen Zellen schon am 4. Tage deutliche retrograde Veranderung. 5. Auf dem Standpunkt stehend, das die Zwischenschaltganglienzelle des Spinalparasympathicus ihren Fortsatz nicht nach der hinteren Wurzel, sondern nur in der Richtung des peripheren Nerven schickt, nahmen wir an, das die kleinen Zellen, die nach der Durchtrennung der hinteren Wurzel unverandert bleiben und nach der des peripheren Nerven sich rasch verandern, als Zwischenschaltganglienzellen des Spinalparasympathicus aufzufassen sind. 6. Genannte kleine Zelle zeigt auch nach der Durchtrennung des Phrenicus, Splanchnicus, sowie nach der Exstirpation des Bauchgrenzstranges rasch retrograde Veranderung. 7. Das Zahlenverhaltnis zwischen den parasympathischen und sensiblen kleinen Zellen stimmt gut mit dem zwischen den parasympathischen und sensiblen kleinmarkhaltigen Fasern uberein. Besonders im Lendengebiet, wo fast alle kleinmarkhaltigen Fasern zum Spinalparasympathicus gehoren, sind fast alle kleinen Zellen (wenigstens 92–93%) den Zwischenschaltganglienzellen zuzurechnen.