Study of the Ca2+-dependent gene expression of EuPrt, an extracellular metalloprotease produced by the psychro-tolerant bacterium Exiguobacterium undae Su-1.

The effect of a Ca2+ ion on the gene expression of an on-demand type of metalloprotease from psychrotrophic Exiguobacterium undae Su-1 (EuPrt) was studied. We first established a modified m m9 medium for strain Su-1 to examine its effect in more detail. Then, when the strain was cultured in m m9 medium and 1.0 m m CaCl2 was added, we detected the mature EuPrt and its precursor proteins via Western blotting analysis and found the relative protease activity and its transcription increased by 50-fold and 7-fold, respectively, at the peak. Furthermore, the intracellular concentration of Ca2+ ions was analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) with other metal ions along the growth of strain Su-1. The intracellular concentration of Ca2+ ion was found to increase as much as 3-fold in response to the addition of an extracellular Ca2+ ions, indicating that euPrt gene expression is regulated by sensing its intracellular concentration.

[Analysis of Symptoms Relieved in Addition to Pain after Administration of Oxycodone or Morphine to Patients with Advanced Cancer Living at Home].

Cancer pain was observed in 131 of 160 patients with advanced cancer living at home. Oxycodone or morphine was administered to patients suffering from cancer pain or fentanyl transdermal patch was switched to morphine. Subsequently, 70 patients were found to have alleviation of symptoms such as nausea/vomiting, dyspnea, abdominal fullness, general fatigue, cough, and urinary urgency in addition to pain. Pain was defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage by the International Association for the Study of Pain. This definition of pain is cited in a lot of books. However, the author was unable to intervene with detailed reports of unpleasant sensations associated with tissue damage. Therefore, here the author reports about an unpleasant sensation caused by tissue damage of cancer that was alleviated by oxycodone or morphine.

[Logical Explanation of Medical Condition for Patients with Terminal Cancer at Home-Retrospective Analysis of 145 Cases].

Frequently, patients with advanced cancer are suffered by various symptoms at the end of life. Furthermore, sometime experience unexpected loss of consciousness(LOC)and/or respiratory arrest. I examined carefully following neck stiffness, LOC, masked face, urinary retention, tonic and/or clonic convulsions, apnea, dysphagia, head rotation, neurogenic pain, hyperthermia, mydriasis 145 patients with advanced cancer. By assuming meningeal carcinomatosis, multifocal neurological findings are logically expected. As a result, before the LOC or respiratory arrest, I was able to logically explain to almost all families of patients, who with suspected neurological signs, expected multifocal neurological signs including LOC or respiratory arrest.

[Influence of the Timing of Cessation of or Withholding Chemotherapy and End-of-Life Care at Home in Patients with Incurable Solid Cancers-An Interim Report of the Results of the Questionnaire Survey among Home Care Physicians in Japan].

BACKGROUND: The study analyzed data obtained using a questionnaire on the potential discriminative characteristics of patients with an incurable solidcancer who receivedor didnot receive palliative chemotherapy during end-of-life care at home. From the standpoint of regional palliative care, we aimed to investigate the influence of the timing of cessation of or withholding chemotherapy andend -of-life care at home in patients with incurable solidcancers. We plannedthe project to obtain scientific evidence about the timing of cessation of or withholding chemotherapy. METHODS: The study included all patients with solidcancers treatedwith or without palliative chemotherapy who diedat home in 2016 in Japan. We distributed postcards of the invitation to participate in the questionnaire survey to more than 2000 home care physicians in Japan. The questionnaires administeredto home care physicians were registeredin website surveys from May to November 2017. The questionnaire data were analyzed using nonparametric methods. RESULTS: We previously obtained information from 576 patients at 170 medical facilities from May to August 2017. As we continue the study, we release an interim report of the questionnaire survey among home care physicians. Of the patients, from the time of diagnosis of the incurable solid cancer, 40% hadreceivedchemotherapy and6 0% hadnot. CONCLUSION: The 60% of patients who didnot undergo chemotherapy since diagnosis were a problem to our projects. However, as we continue the questionnaire survey, we would like to analyze the data from the returned questionnaires.

[The Influence of the Timing of Ceasing or Withholding Chemotherapy in Home-Based End-of-Life Care in Patients with Incurable Solid Cancer - Release of an Interim Report of the Questionnaire Survey to Home Care Doctors in Japan].

BACKGROUND: The study analyzes a questionnaire on the potential discriminative characteristics of patients with incurable solidcancer, who either receivedor didnot receive palliative chemotherapy while receiving home-basedend -of-life care. From the standpoint of regional palliative care, we sought to investigate the influence of the timing of when chemotherapy was ceasedor withheldin home-basedend -of-life care in patients with incurable solidcancer. We plannedthe project to obtain scientific evidence about the timing of ceasing or withholding chemotherapy. PATIENTS AND METHODS: The study includes all patients with solidcancer treatedwith or without palliative chemotherapy andwho diedat home in 2016 in Japan. We delivereda postcardof invitation to participate in the questionnaire to more than 2,000 home care doctors in Japan. The questionnaires were registeredas online surveys from May to November 2017. The questionnaire data were analyzed using nonparametric methods. RESULTS: We obtained information from 576 patients at 170 medical facilities from May to August 2017, but the study is currently ongoing; hence, we have released an interim report of the questionnaire results. Among the patients, 40%receivedchemotherapy and 60%didnot since the time of the first incurable solidcancer diagnosis. CONCLUSION: The majority 60% of patients not receiving chemotherapy was a setback to our project. However, as the questionnaire survey continues, we wouldlike to analyze these data after collecting more results.

Caenibacillus caldisaponilyticus gen. nov., sp. nov., a thermophilic, spore-forming and phospholipid-degrading bacterium isolated from acidulocompost.

A thermophilic and phospholipid-degrading bacterium, designated strain B157T, was isolated from acidulocompost, a garbage compost processed under acidic conditions at moderately high temperature. The organism was Gram-stain-positive, aerobic, spore-forming and rod-shaped. Growth was observed to occur at 40-65 °C and pH 4.8-8.1 (optimum growth: 50-60 °C, pH 6.2). The strain was catalase- and oxidase-positive. The cell wall contained meso-diaminopimelic acid, alanine, glutamic acid and galactose. The predominant respiratory quinone was menaquinone-7 (MK-7) and the major fatty acids were anteiso-C17 : 0 and iso-C17 : 0. Comparative 16S rRNA gene sequence analysis showed that strain B157T was related most closely to Tuberibacillus calidus 607T (94.8 % identity), and the phylogenetic analysis revealed that it belonged to the family Sporolactobacillaceae. The DNA G+C content was determined as 51.8 mol%. In spite of many similarities with the type strains of members of the family Sporolactobacillaceae, genotypic analyses suggest that strain B157T represents a novel species of a new genus, Caenibacilluscaldisaponilyticus gen. nov., sp. nov. The type strain of Caenibacilluscaldisaponilyticus is B157T (=NBRC 111400T=DSM 101100T).

Bacterial membrane vesicles (MVs): novel tools as nature- and nano-carriers for immunogenic antigen, enzyme support, and drug delivery.

It is becoming well-known that bacterial cells produce membrane vesicles (MVs) from the cell surface in a budding manner, whereas the detailed mechanisms of MV biogenesis remain unclear. MVs are not authentic cells, since they are observed to be between 20 and 300 nm in size but have a structure close to the subcellular compartments. In a sense, the structure of MVs containing biogenic and cellular substances and their behavior look similar to those of viruses. Due to these scientific facts, several potent applications employing MVs as a promising tool have been proposed and reported. This review introduces a few outstanding examples for promising applications of MVs to biotechnology.

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae.

SNQ2 was identified as a caffeine-resistance gene by screening a genomic library of Saccharomyces cerevisiae in a multicopy vector YEp24. SNQ2 encodes an ATP-binding cassette transporter and is highly homologous to PDR5. Multicopy of PDR5 also conferred resistance to caffeine, while its resistance was smaller than that of SNQ2. Residual caffeine contents were analyzed after transiently exposing cells to caffeine. The ratios of caffeine contents were 21.3 ± 8.8% (YEp24-SNQ2) and 81.9 ± 8.7% (YEp24-PDR5) relative to control (YEp24, 100%). In addition, multicopies of SNQ2 or PDR5 conferred resistance to rhodamine 6G (R6G), which was widely used as a substrate for transport assay. R6G was exported by both transporters, and their efflux activities were inhibited by caffeine with half-maximal inhibitory concentrations of 5.3 ± 1.9 (YEp24-SNQ2) and 17.2 ± 9.6 mM (YEp24-PDR5). These results demonstrate that Snq2p is a more functional transporter of caffeine than Pdr5p in yeast cells.

Extraordinary denaturant tolerance of keratinolytic protease complex assemblies produced by Meiothermus ruber H328.

A moderately thermophilic bacterial strain, Meiothermus ruber H328, can efficiently solubilize intact chicken feathers by aerobic cultivation at 55 °C for 6 days. The keratinolytic proteases extracellularly secreted by the strain were partially purified by an ultrafiltration system and a size-exclusion column chromatography, and thus were found to be two different sizes of macromolecules with an extremely high molecular mass like the sizes of virus and DNA (peak 1 fraction) and with a molecular mass of larger than 500 kDa (peak 2 fraction). They formed protein complex assemblies that were composed of multiple but different proteins. The peak 1 fraction showed more thermophilic characteristics than did the peak 2 fraction in temperature dependence and thermal stability. By contrast, they comparably showed extraordinary resistance to powerful denaturants, SDS at 30 % (w/v) and organic solvents (methanol, ethanol, acetonitrile, acetone, and chloroform) at 40 % (v/v) at 60 °C for 30 min. The extraordinary denaturant tolerance and the large molecular size of the keratinolytic protease complex assemblies suggest the possibility that those may be lipophilic and have the structure of partial membrane fractions, or membrane vesicles, which are exfoliated from the outer membrane of the cells.

Meiothermus ruber H328 enhances the production of membrane vesicles for feather degradation.

The thermophilic bacterium Meiothermus ruber H328 aggressively degrades chicken feathers. When feathers were added to culture medium, the cells significantly exfoliated membrane vesicles from the outer membrane as observed by electron microscopy of ultrathin sections. This is the first report of membrane vesicle production associated with keratinolytic activity by Meiothermus sp.

Combination of four bacterial strains isolated from Yamahai-shubo in traditional Japanese sake brewing.

This study investigated the interactions of four bacteria strains isolated from Yamahai-shubo, the source of yeast used to produce a Japanese traditional rice wine, Yamahai-shikomi sake. The bacterial strains were nitrate-reducing Pseudomonas sp. 61-02, Leuconostoc mesenteroides LM-1, Lactiplantibacillus plantarum LP-2, and Latilactobacillus sakei LS-4. We examined fermentation factors for Yamahai-shubo and Yamahai-shikomi sake samples to compare the suitability of their bacterial combination (16 variations). As a result of principal component analysis, we found that two major groups were formed; one containing strain LP-2 and the other containing strain LS-4, and that strains LP-2 and LS-4 were important in the Yamahai-shikomi sake in the presence of strains 61-02 and LM-1. Then, we investigated the effects of strains LP-2 and LS-4 on the concentration of organic acids (pyruvic acid, citric acid, succinic acid, malic acid, and lactic acid) in Yamahai-shikomi sake. Only in lactic acid, a tendency to decrease with a smaller proportion of LS-4 strains in Yamahai-shubo was observed. Subsequently, their effect on the concentration of diacetyl, crucial for aroma, was investigated between the LP-2 and LS-4 strains. The sample prepared in the absence of strain LS-4 exhibited the lowest concentration of diacetyl. This result was supported by the statistical analysis for the sensory scores performed for aroma of each Yamahai-shikomi sake sample. In conclusion, strain LP-2 plays a more significant role in improving Yamahai-shikomi sake quality with strains LM-1 and 61-02 rather than strain LS-4 in Yamahai-shubo preparation and Yamahai-shikomi sake brewing.

Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1.

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Šat 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as `Kibo') at the International Space Station (ISS). The crystal belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 87.64, c = 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.

An on-demand metalloprotease from psychro-tolerant Exiguobacterium undae Su-1, the activity and stability of which are controlled by the Ca2+ concentration.

We reported an on-demand type of metalloprotease from Exiguobacterium undae Su-1. Although this species of bacterium is known to inhabit the permafrost, there are no reports on either strong proteases or peptidases. We found that Su-1 protease is superior to commercially available proteases in proteolytic activity in a lower to normal range of temperature (10-50 °C) as well as in rapid inactivation heat-dependently on the Ca2+ concentration. These characteristics meet well with the demands from food processing and manufacturing. Biochemical investigations of the purified enzyme and protein structural analysis after gene cloning confirmed that Su-1 protease conserved high identity in its primary sequence with thermophilic proteases of the M4 family. On the other hand, its flexibility was enhanced when one Ca2+ binding site was lost and by replacement for proline and isoleucine residues.

The exquisite structure and reaction mechanism of bacterial Pz-peptidase A toward collagenous peptides: X-ray crystallographic structure analysis of PZ-peptidase a reveals differences from mammalian thimet oligopeptidase.

Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80-2.00 Å resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.

Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328.

The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain ∆degP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain ∆degP exhibited smooth growth but a lower level of turbidity at 60 °C although there was no difference in growth at 55 °C between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60 °C increases MV in the mutant strain ∆degP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60 °C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60 °C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.

Molecular characterization of a prolyl endopeptidase from a feather-degrading thermophile Meiothermus ruber H328.

Prolyl endopeptidase from an aerobic and Gram-negative thermophile Meiothermus ruber H328 (MrPEP) was purified in native and recombinant forms, but both preparations had comparable characteristics. Production of the native MrPEP was increased 10-fold by adding intact chicken feathers. The gene for MrPEP (mrH_2860) was cloned from the genome of strain H328 and found to have no signal sequence at the N-terminus. MrPEP is composed of two major domains: the ß-propeller domain and the peptidase domain with a typical active site motif and catalytic triad. Based on extensive investigations with different types of peptide substrates and FRETS-25Xaa libraries, MrPEP showed strict preferences for Pro residue at the P1 position but broader preferences at the P2 and P3 positions in substrate specificity with stronger affinity for residues at the P3 position of substrate peptides that are longer than four residues in length. In conclusion, the molecular characterization of MrPEP resembles its animal counterparts more closely than bacterial counterparts in function and structure.

Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic Meiothermus ruber H328.

The sustainable and practical degradation of intact chicken feathers by a newly isolated thermophilic bacterium Meiothermus ruber H328 was presented with extensive data. Aerobic cultivation with moderately thermophilic strain H328 at 55 degrees C for 6 days led to the apparently complete decay of the truly intact feathers and provided 1.89 mmol free amino acids and 7.32 mmol acid-hydrolyzed amino acids from 50 ml of culture containing 3% (w/v) intact chicken feathers. The amino acid components in the soluble fraction of the culture conspicuously agreed with those calculated from the intact feathers. This demonstrated that more than 55% of total keratin proteins were solubilized from the intact chicken feathers into the culture in the forms of free amino acid and/or soluble oligopeptide, and most of them are directly derived from the intact feathers by proteolytic digestion. Feather degradation by strain H328 surpasses that by any other microorganisms with regard to degradation efficiency, absence of requirement for pretreatment of the feathers, and product fidelity in the amino acid component. Furthermore, the culture containing the degradative products from the intact feathers was subjected to matrix-assisted laser desorption ionization mass spectrometry-time-of-flight analysis, and it was revealed that the molecular masses of the solubilized products, oligopeptides, were less than 1,000. This result allows us to investigate the bioactivities of oligopeptides derived from the degradation of chicken feathers by cultivation with strain H328 as well as the production of amino acids for feedstuffs.

Decomposition of intact chicken feathers by a thermophile in combination with an acidulocomposting garbage-treatment process.

In order to develop a practical method for the decomposition of intact chicken feathers, a moderate thermophile strain, Meiothermus ruber H328, having strong keratinolytic activity, was used in a bio-type garbage-treatment machine working with an acidulocomposting process. The addition of strain H328 cells (15 g) combined with acidulocomposting in the garbage machine resulted in 70% degradation of intact chicken feathers (30 g) within 14 d. This degradation efficiency is comparable to a previous result employing the strain as a single bacterium in flask culture, and it indicates that strain H328 can promote intact feather degradation activity in a garbage machine currently on the market.

Effect of Nitric Oxide on the Oxygen Metabolism and Growth of E. faecalis.

Gastro-intestinal mucosal cells have a potent mechanism to eliminate a variety of pathogens using enzymes that generate reactive oxygen species and/or nitric oxide (NO). However, a large number of bacteria survive in the intestine of human subjects. Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that survives not only in the intestinal lumen but also within macrophages generating NO. It has been reported that E. faecalis generated the superoxide radical (O(2) (-)). To elucidate the role of O(2) (-) and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O(2) (-) and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 micromol O(2) (-)/min/10(8) cells in a glucose-dependent manner as determined using the cytochrome c reduction method. The presence of NOC12, an NO donor, strongly inhibited the growth of E. faecalis without affecting in the oxygen consumption. However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells. Western blotting analysis revealed that the NOC12-treated E. faecalis revealed a large amount of nitrotyrosine-posititive proteins; the amounts of the modified proteins were higher in cytosol than in membranes. These observations suggested that O(2) (-) generated by E. faecalis reacted with NO to form peroxinitrite (ONOO(-)) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth. Since E. faecalis survives even within macrophages expressing NO synthase, similar metabolism of O(2) (-) and NO may occur in and around phagocytized macrophages.

Purification and gene cloning of alpha-methylserine aldolase from Ralstonia sp. strain AJ110405 and application of the enzyme in the synthesis of alpha-methyl-L-serine.

By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.