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To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (CO

Cancer consists of heterogeneous cells that contain a small population of cells that possess stem cell properties; these cells, referred to as cancer stem cells (CSCs) or tumor-initiating cells, are involved in tumor progression and metastasis. Using a sphere-forming assay, canine mammary CSCs were found to be similar to human breast CSCs. Metabolic reprogramming has been recognized as a hallmark of various cancers. However, the significance of cellular metabolism in CSCs remains unclear. The aim of this study was to define the metabolic characteristics of CSCs derived from canine mammary tumors and gain an understanding of the maintenance of stemness. We identified metabolite profiles of canine mammary adenocarcinoma cell lines using gas chromatography-mass spectrometry. Metabolites were extracted from both adherent and sphere-forming cells derived from three cell lines. Sphere-forming cells were separated from adherent cells using an orthogonal, partial least-squares discriminant analysis. Sphere-forming cells were found to contain high levels of the amino acids alanine, glycine and proline compared with adherent cells. They also had high levels of palmitoleate, palmitate and dihomo-gamma-linolenic acid compared with adherent cells. In a sphere-forming assay, palmitate increased the number of spheres for all cell lines. These results indicate that the sphere-forming cells derived from canine mammary adenocarcinoma cell lines have specific metabolic profiles that may be useful for the development of CSC-specific therapies targeting metabolic pathways and potential stemness biomarkers; these results also clarify the maintenance of stemness in canine mammary CSCs.

Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T=DSM 46748T).

A previously undescribed rapidly growing, non-pigmented mycobacterium was identified based on biochemical and nucleic acid analyses, as well as growth characteristics. Seven isolates were cultured from samples collected from five thread-sail filefish (Stephanolepis cirrhifer) and two farmed black scraper (Thamnaconus modestus). Bacterial growth occurred at 15–35 °C on Middlebrook 7H11 agar. The bacteria were positive for catalase activity at 68 °C and urease activity, intermediate for iron uptake, and negative for Tween 80 hydrolysis, nitrate reduction, semi-quantitative catalase activity and arylsulfatase activity at day 3. No growth was observed on Middlebrook 7H11 agar supplemented with picric acid, and very little growth was observed in the presence of 5 % NaCl. α- and α′-mycolates were identified in the cell walls, and a unique profile of the fatty acid methyl esters and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of the protein and cell-wall lipids were acquired. Sequence analysis revealed that the seven isolates shared identical sequences for the 16S rRNA, rpoB, hsp65, recA and sodA genes. Phylogenetic analysis of the five gene sequences confirmed that the isolates were unique, but closely related to Mycobacterium chelonae . Antibiotic susceptibility testing revealed the minimum inhibitory concentration (MIC) of clarithromycin against this novel species was

Several phytotoxic metabolites, including a novel compound chaetoglobosin O, were isolated from Cylindrocladium floridanum Sobers et Seymore. The structure of chaetoglobosin O, including its absolute configuration, was determined by a spectroscopic analysis and chemical correlation. Purified chaetoglobosins A, C, and O showed potent growth-inhibition activity against alfalfa seedlings.

The in vitro cultured liverwort Jungermannia subulata produces the unique molecule subulatin. In this study, we examined the incorporation of [1-13C] and [1,2-13C2] glucose, [2-13C] arabinose, [2-13C] caffeic acid, and [1-13C] phenylalanine into subulatin. The trilobatinoic acid C unit of subulatin incorporated 13C atoms from [1-13C] and [1,2-13C2] glucose and from [2-13C] arabinose but not from any other of the other precursors. Based on these results and labeling patterns, the trilobatinoic acid C unit of subulatin appears to be biosynthesized from arabinose-5-phosphate and phosphoenolpyruvate.

Arthrobacter sp. K-1 β-fructofuranosidase (ArFFase), a glycoside hydrolase family 68 enzyme, catalyzes the hydrolysis and transfructosylation of sucrose. ArFFase is useful for producing a sweetener, lactosucrose (4(G)-β-D-galactosylsucrose). The primary structure of ArFFase is homologous to those of levansucrases, although ArFFase catalyzes mostly hydrolysis when incubated with sucrose alone, even at high concentration. Here, we determined the crystal structure of ArFFase in unliganded form and complexed with fructose. ArFFase consisted of a five-bladed β-propeller fold as observed in levansucrases. The structure of ArFFase was most similar to that of Gluconacetobacter diazotrophicus levansucrase (GdLev). The structure of the catalytic cleft of ArFFase was also highly homologous to that of GdLev. However, two amino acid residues, Tyr232 and Pro442 in ArFFase, were not conserved between them. A tunnel observed at the bottom of the catalytic cleft of ArFFase may serve as a water drain or its reservoir.

Simple liquid chromatography-mass spectrometry (LC-MS) was applied to non-targeted metabolic analyses to discover new metabolic markers in animal plasma. Principle component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) were used to analyse LC-MS multivariate data. PCA clearly generated two separate clusters for artificially induced diabetic mice and healthy control mice. PLS-DA of time-course changes in plasma metabolites of chicks after feeding generated three clusters (pre- and immediately after feeding, 0.5-3 h after feeding and 4 h after feeding). Two separate clusters were also generated for plasma metabolites of pregnant Angus heifers with differing live-weight change profiles (gaining or losing). The accompanying PLS-DA loading plot detailed the metabolites that contribute the most to the cluster separation. In each case, the same highly hydrophilic metabolite was strongly correlated to the group separation. The metabolite was identified as betaine by LC-MS/MS. This result indicates that betaine and its metabolic precursor, choline, may be useful biomarkers to evaluate the nutritional and metabolic status of animals.

Circulating levels of monosaccharides can act as a reflection of systemic glucose/ energy metabolism. Characteristic changes observed in these levels can be seen in patients with diabetes and other metabolic disorders. There have been a few reports describing the significance of mannose metabolism as an energy source under physiological and pathological conditions. However, the relationship between circulating levels of mannose and the pathophysiology of diabetes mellitus are unknown in dogs. This study examined circulating levels of mannose between healthy control and diabetic dogs and evaluated the clinical significance of mannose levels in dogs. Diabetic dogs demonstrated a higher circulating level of mannose in comparison to normal healthy control dogs. Plasma mannose was positively correlated with plasma glucose and fructosamine, respectively. Interestingly, plasma mannose levels were affected by plasma insulin levels. In the context of feeding and glucose tolerance tests, plasma mannose levels responded to changes in circulating insulin levels. Circulating plasma mannose levels decreased after feeding in both control and diabetic animals in spite of observed insulin level differences. However, when glucose tolerance tests were given, a positive correlation between mannose levels and insulin levels was observed. Therefore, plasma mannose levels obtained via glucose tolerance testing may be used as a new diagnostic method for evaluating insulin resistance or deficiency in diabetic dogs.

New clerodane-type diterpenes, designated as parvitexins A (1)-E (5), were isolated from the in vitro-cultured liverwort, Scapania parvitexta. These compounds were determined to be monoacetylated clerodane-type diterpenes based on spectroscopic evidence.

In dogs, hyperadrenocorticism (HAC) is associated with insulin resistance and diabetes does progress with HAC. There are significant differences in the transcriptomic and proteomic patterns of activated T cells, which parallel the findings in muscle tissues. The aim of this study was to assess how glucocorticoids affect intracellular metabolites in canine peripheral blood mononuclear cells (CnPBMCs) using dexamethasone. A total of 96 metabolites were identified by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). After incubation with dexamethasone, the metabolites glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, sedoheptulose 7-phosphate and acetyl-CoA were significantly increased. However, ATP, CTP, dATP, pyruvic acid and NADP(+) were significantly decreased. These results show that a glucocorticoid reduces the catabolic reaction of glucose and accordingly decreases the glucose requirements of CnPBMCs.

Tetracenomycin F2 cyclase (tcmI gene product), catalyzes an aromatic rearrangement in the biosynthetic pathway for tetracenomycin C in Streptomyces glaucescens. The x-ray structure of this small enzyme has been determined to 1.9-A resolution together with an analysis of site-directed mutants of potential catalytic residues. The protein exhibits a dimeric βαβ ferredoxin-like fold that utilizes strand swapping between subunits in its assembly. The fold is dominated by four strands of antiparallel sheet and a layer of α-helices, which creates a cavity that is proposed to be the active site. This type of secondary structural arrangement has been previously observed in polyketide monooxygenases and suggests an evolutionary relationship between enzymes that catalyze adjacent steps in these biosynthetic pathways. Mutational analysis of all of the obvious catalytic bases within the active site suggests that the enzyme functions to steer the chemical outcome of the cyclization rather than providing a specific catalytic group. Together, the structure and functional analysis provide insight into the structural framework necessary to perform the complex rearrangements catalyzed by this class of polyketide cyclases.

The syntheses of prosolanapyrones I (6) and II (7) via the aldol reactions of pyrone and dienal segments have been achieved in five steps in 31% overall yield for 6 and seven steps in 5% overall yield for 7. An improved synthetic route starting from vinylpyrone 27 provided 7 in 11 steps in 12% overall yield. The enzymatic Diels−Alder reaction of 7 affords (−)-solanapyrone A (1) with high enantioselectivity and with good exo-selectivity, which is difficult to attain by chemical methods. In addition, a crude enzyme preparation from Alternaria solani has been used to perform a kinetic resolution of (±)-3.

A cell-free extract from the calli of the liverwort Heteroscyphus planus catalyzes the divalent metal ion-dependent conversion of (2Z, 6E)-farnesyl diphosphate (FPP) into (-)-γ-cadinene and (+)-germacrene D, while it specifically converts (2E, 6E)-FPP into (+)-cubenene and (+)-epicubenol. The 1,3-hydride shift in the formation of (-)-γ-cadinene has been determined by conversion of (2Z, 6E)-[1,1-2H2]-FPP into (-)-γ-cadinene which was shown by GLC–MS and 2H NMR spectroscopy to be labelled at the C-11 position. These findings suggest that (-)-γ-cadinene is directly formed from 2Z, 6E-FPP by intramolecular electrophilic attack of the primary carbocation on the C-10 position of FPP. (+)-Cubenene synthase and (-)-γ-cadinene synthase has been purified by fractionation with ammonium sulfate, gel filtration on Sephacryl S-200 HR and anion exchange chromatography on DEAE-Sepharose CL-6B. Separation of (-)-γ-cadinene synthase from (+)-cubenene synthase is facilitated by a change in the elution behaviour of enzymes during anion exchange chromatography. All the evidence strongly suggests that the activities of (+)-cubenene synthase and (+)-epicubenol synthase are dual functions of the same enzyme.

The establishment of a classification system for domestic animals on consumed feed stuff is thought to be important from both a hygiene and market point of view. We collected plasma samples of Romney lambs (Ovis aries) which were fed one of the following: a herb-clover mix (n=10) which included chicory, red clover, white clover and plantain; a plant-grass mix (n=10) which included plantain, ryegrass and white clover; or a grass mix (n=10) which included ryegrass and white clover. A total of 20 elements in plasma samples obtained from the lambs were analyzed using inductively coupled plasma mass spectrometry. The data were then analyzed by principal component analysis. The lambs were divided into three groups on a score plot depending on the different feed conditions. Furthermore, discriminant analyses of the elements were examined, using linear discriminant analysis with forward stepwise regression. This discriminant function correctly classified the samples from each group. The accuracy of classification of each group, as shown by 10-fold cross-validation, proved the effectiveness of the established discriminant function. It is concluded that using linear discriminant analysis might be a useful tool for the validation of elements from plasma in lambs grown in different conditions.

A cell-free extract from the calli of the liverwort Heteroscyphus planus catalyzes the divalent metal ion-dependent conversion of (2Z, 6E)-farnesyl diphosphate (FPP) into (-)-γ-cadinene and (+)-germacrene D, while it specifically converts (2E, 6E)-FPP into (+)-cubenene and (+)-epicubenol. The 1,3-hydride shift in the formation of (-)-γ-cadinene has been determined by conversion of (2Z, 6E)-[1,1-2H2]-FPP into (-)-γ-cadinene which was shown by GLC–MS and 2H NMR spectroscopy to be labelled at the C-11 position. These findings suggest that (-)-γ-cadinene is directly formed from 2Z, 6E-FPP by intramolecular electrophilic attack of the primary carbocation on the C-10 position of FPP. (+)-Cubenene synthase and (-)-γ-cadinene synthase has been purified by fractionation with ammonium sulfate, gel filtration on Sephacryl S-200 HR and anion exchange chromatography on DEAE-Sepharose CL-6B. Separation of (-)-γ-cadinene synthase from (+)-cubenene synthase is facilitated by a change in the elution behaviour of enzymes during anion exchange chromatography. All the evidence strongly suggests that the activities of (+)-cubenene synthase and (+)-epicubenol synthase are dual functions of the same enzyme.

Advanced intermediates, prosolanapyrones I (6) and II (7) have been synthesized in deuterium labelled form and administered to cultures of Alternaria solani. Incorporation of [17,17,18,18,18-2H5]prosolanapyrone I (6b) afforded solanapyrones A (1) labelled at C-17 and C-18 with the expected integration in its 2H NMR spectrum. Subsequently, [2,3,17,18,18,18-2H6]prosolanapyrone II (7a) was incorporated into solanapyrone A labelled, as expected, at C-2, C-3, C-17 and C-18. These results strongly support the involvement of a Diels–Alder reaction in the biosynthesis of solanapyrones. This is the first example of intact incorporation of diene-dienophile precursors into natural [4 + 2] adducts.

In vitro cultured gametophytes and suspension cells of the liverwort, Heteroscyphus planus , were assayed for the accumulation of mono- and sesquiterpenes. (+)-α-Pinene, sesquiterpene hydrocarbons and oxygenated sesquiterpenes including epicubenol and four cadinane-type sesquiterpenes were detected by GLC and GC-MS analyses. (1 S ,4 R )-7-Methoxycalamenene, (1 S )-7-methoxy-1,2-dihydrocadalene,7-methoxycadalene and (1 S ,4 R )-7-hydroxycalamenene were isolated and identified by spectral data (NMR, MS, UV and IR) and chemical correlations.

The first natural Diels-Alderase, solanapyrone synthase, was purified 1,630-fold from a crude extract. The 41-kDa protein on SDS-polyacrylamide gel electrophoresis was identified as truncated solanapyrone synthase, and its N-terminal amino acid sequence was found to be QETQNLNNFLESNAINP.

Tetracenomycin F2 cyclase (tcmI gene product), catalyzes an aromatic rearrangement in the biosynthetic pathway for tetracenomycin C in Streptomyces glaucescens. The x-ray structure of this small enzyme has been determined to 1.9-A resolution together with an analysis of site-directed mutants of potential catalytic residues. The protein exhibits a dimeric betaalphabeta ferredoxin-like fold that utilizes strand swapping between subunits in its assembly. The fold is dominated by four strands of antiparallel sheet and a layer of alpha-helices, which creates a cavity that is proposed to be the active site. This type of secondary structural arrangement has been previously observed in polyketide monooxygenases and suggests an evolutionary relationship between enzymes that catalyze adjacent steps in these biosynthetic pathways. Mutational analysis of all of the obvious catalytic bases within the active site suggests that the enzyme functions to steer the chemical outcome of the cyclization rather than providing a specific catalytic group. Together, the structure and functional analysis provide insight into the structural framework necessary to perform the complex rearrangements catalyzed by this class of polyketide cyclases.

The crude enzyme from Alternaria solani is able to catalyse the [4 + 2] cycloaddition of prosolanapyrone III 6 to the exo adduct solanapyrone A 1 whose optical purity is estimated as 92 ± 8% e.e. by HPLC analysis monitored using a CD spectrometer; this enzyme also catalyses the oxidation and [4 + 2] cycloaddition of prosolanapyrone II 5 to 1 with 99 ± 4% e.e.

(1993). Occurrence of (+)-α-Cedrene in Larix leptolepis Callus. Bioscience, Biotechnology, and Biochemistry: Vol. 57, No. 6, pp. 1022-1023.

Advanced intermediates, prosolanapyrones I (6) and II (7) have been synthesized in deuterium labelled form and administered to cultures of Alternaria solani. Incorporation of [17,17,18,18,18-2H5]prosolanapyrone I (6b) afforded solanapyrones A (1) labelled at C-17 and C-18 with the expected integration in its 2H NMR spectrum. Subsequently, [2,3,17,18,18,18-2H6]prosolanapyrone II (7a) was incorporated into solanapyrone A labelled, as expected, at C-2, C-3, C-17 and C-18. These results strongly support the involvement of a Diels–Alder reaction in the biosynthesis of solanapyrones. This is the first example of intact incorporation of diene-dienophile precursors into natural [4 + 2] adducts.