Differences in susceptibility of cyanobacteria species to lytic volatile organic compounds and influence on seasonal succession.

Cyanobacteria produce numerous volatile organic compounds (VOCs) that show a lytic activity against other cyanobacteria. We found the lytic phenomenon under natural conditions and during densification experiments, and also observed the species change of the cyanobacteria during the lysis processes, in which Microcystis finally became dominant. The species change of the cyanobacteria was strongly suggested to depend on the susceptibility of the cyanobacteria toward the VOCs. To verify this suggestion, the susceptibility of the species was evaluated by the minimal inhibitory concentration (MIC) using axenic cyanobacterial strains against ß-cyclocitral, its oxidation products and ß-ionone with the aid of log D. It was found that the difference depended on the susceptibility of the cyanobacteria toward the VOCs, in which ß-cyclocitral played a crucial role and Microcystis had a significantly protective ability compared to the other cyanobacteria. In addition, the species change of cyanobacteria was consistent with the cyanobacterial seasonal succession in Lakes Sagami and Tsukui, based on data that had been accumulated for 10 years. Conventionally, although this phenomenon could be explained by nutrient availability or the physical structure of the environment, the results of this study revealed that it was controlled by the VOCs, particularly ß-cyclocitral produced by the cyanobacteria.

[Analysis of Dieldrin and DTTB in Textiles].

Standard analytical methods for the detection of dieldrin and 4,6-dichloro-7-(2,4,5-trichlorophenoxy)-2-trifluoromethylbenzimidazole (DTTB) in textiles, which are regulated by Japanese law ("Act on the Control of Household Products Containing Harmful Substances"), have been in place for more than 30 years. In this study, we developed an improved analytical method, based on GC-MS, that uses safe reagents and can simultaneously detect dieldrin and DTTB analytes. In the standard (existing) analytical method, dimethyl sulfate, which is a potential carcinogen, is used to derivatize DTTB. In the developed method, phenyltrimethylammonium hydroxide, as an alternative reagent, was used to derivatize DTTB in good results. Dieldrin and the derivatized DTTBs gave highly linear calibration curves when analyzed by GC-MS. Moreover, we found that both analytes are adequately extracted from textiles by refluxing in hydrochloric acid and methanol. Furthermore, we established a purification method using the Bond Elut PRS column that effectively removed interfering substances in woolen products. Finally, we developed an improved analysis method by combining the above-mentioned techniques; the developed method exhibited a recovery rate of 94-104% and a relative standard deviation of less than 7% for both analytes. In addition, the limits of quantitation (dieldrin: 1.3 µg/g, DTTB: 0.72 µg/g) were sufficiently lower than the Japanese regulatory value of 30 µg/g.

Analytical Technique Optimization on the Detection of ß-cyclocitral in Microcystis Species.

ß-Cyclocitral, specifically produced by Microcystis, is one of the volatile organic compounds (VOCs) derived from cyanobacteria and has a lytic activity. It is postulated that ß-cyclocitral is a key compound for regulating the occurrence of cyanobacteria and related microorganisms in an aquatic environment. ß-Cyclocitral is sensitively detected when a high density of the cells is achieved from late summer to autumn. Moreover, it is expected to be involved in changes in the species composition of cyanobacteria in a lake. Although several analysis methods for ß-cyclocitral have already been reported, ß-cyclocitral could be detected using only solid phase micro-extraction (SPME), whereas it could not be found at all using the solvent extraction method in a previous study. In this study, we investigated why ß-cyclocitral was detected using only SPME GC/MS. Particularly, three operations in SPME, i.e., extraction temperature, sample stirring rate, and the effect of salt, were examined for the production of ß-cyclocitral. Among these, heating (60 °C) was critical for the ß-cyclocitral formation. Furthermore, acidification with a 1-h storage was more effective than heating when comparing the obtained amounts. The present results indicated that ß-cyclocitral did not exist as the intact form in cells, because it was formed by heating or acidification of the resulting intermediates during the analysis by SPME. The obtained results would be helpful to understand the formation and role of ß-cyclocitral in an aquatic environment.

Complete Genome Sequence of a Microcystin-Degrading Bacterium, Sphingosinicella microcystinivorans Strain B-9.

Sphingosinicella microcystinivorans strain B-9 has the ability to degrade cyanobacterial hepatotoxic cyclic peptides, microcystins, and nodularins. This is the first report of the complete genome sequence of the microcystin-degrading bacterium.

Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B-9.

Strain B-9, which has a 99% similarity to Sphingosinicella microcystinivorans strain Y2, is a Gram-negative bacterium with potential for use in the degradation of microcystin-related compounds and nodularin. We attempted to extend the application area of strain B-9 and applied it to mycotoxins produced by fungi. Among the tested mycotoxins, only ochratoxin A was completely hydrolyzed to provide the constituents ochratoxin α and l-phenylalanine, and levels of fumonisin B1 gradually decreased after 96 h. However, although drugs including antibiotics released into the aquatic environment were applied for microbial degradation using strain B-9, no degradation occurred. These results suggest that strain B-9 can only degrade amino acid-containing compounds. As expected, the tested compounds with amide and ester bonds, such as 3,4-dimethyl hippuric acid and 4-benzyl aspartate, were readily hydrolyzed by strain B-9, although the sulfonamides remained unchanged. The ester compounds were characteristically and rapidly hydrolyzed as soon as they came into contact with strain B-9. Furthermore, the degradation of amide and ester compounds with amino acids was not inhibited by the addition of ethylenediaminetetraacetic acid (EDTA), indicating that the responsible enzyme was not MlrC. These results suggest that strain B-9 possesses an additional hydrolytic enzyme that should be designated as MlrE, as well as an esterase.

Characteristic oxidation behavior of ß-cyclocitral from the cyanobacterium Microcystis.

The cyanobacterium Microcystis produces volatile organic compounds such as ß-cyclocitral and 3-methyl-1-butanol. The lysis of cyanobacteria involving the blue color formation has been occasionally observed in a natural environment. In this study, we focused on the oxidation behavior of ß-cyclocitral that contributed to the blue color formation in a natural environment and compared ß-cyclocitral with a structurally related compound concerning its oxidation, acidification, and lytic behavior. The oxidation products of ß-cyclocitral were identified by the addition of ß-cyclocitral in water, in which 2,2,6-trimethylcyclohex-1-ene-1-yl formate and 2,2,6-trimethylcyclohexanone were structurally characterized. That is, ß-cyclocitral was easily oxidized to produce the corresponding carboxylic acid and the enol ester in water without an oxidizing reagent, suggesting that this oxidation proceeded according to the Baeyer-Villiger oxidation. The oxidation behavior of ß-cyclocitral in a laboratory was different from that in the natural environment, in which 2,2,6- trimethylcyclohexanone was detected at the highest amount in the natural environment, whereas the highest amount in the laboratory was ß-cyclocitric acid. A comparison of ß-cyclocitral with structurally similar aldehydes concerning the lytic behavior of a Microcystis strain and the acidification process indicated that only ß-cyclocitral was easily oxidized. Furthermore, it was found that a blue color formation occurred between pH 5.5 and 6.5, suggesting that chlorophyll a and ß-carotene are unstable and decomposed, whereas phycocyanin was stable to some extent in this range. The obtained results of the characteristic oxidation behavior of ß-cyclocitral would contribute to a better understanding of the cyanobacterial life cycle.

Cyanobacterial blue color formation during lysis under natural conditions.

Cyanobacteria produce numerous volatile organic compounds (VOCs), such as ß-cyclocitral, geosmin, and 2-methylisoborneol, which show lytic activity against cyanobacteria. Among these compounds, only ß-cyclocitral causes a characteristic color change from green to blue (blue color formation) in the culture broth during the lysis process. In August 2008 and September 2010, the lysis of cyanobacteria involving blue color formation was observed at Lake Tsukui in northern Kanagawa Prefecture, Japan. We collected lake water containing the cyanobacteria and investigated the VOCs, such as ß-cyclocitral, ß-ionone, 1-propanol, 3-methyl-1-butanol, and 2-phenylethanol, as well as the number of cyanobacterial cells and their damage and pH changes. As a result, the following results were confirmed: the detection of several VOCs, including ß-cyclocitral and its oxidation product, 2,2,6-trimethylcyclohexene-1-carboxylic acid; the identification of phycocyanin based on its visible spectrum; the lower pH (6.7 and 5.4) of the lysed samples; and characteristic morphological change in the damaged cyanobacterial cells. We also encountered the same phenomenon on 6 September 2013 in Lake Sagami in northern Kanagawa Prefecture and obtained almost the same results, such as blue color formation, decreasing pH, damaged cells, and detection of VOCs, including the oxidation products of ß-cyclocitral. ß-Cyclocitral derived from Microcystis has lytic activity against Microcystis itself but has stronger inhibitory activity against other cyanobacteria and algae, suggesting that the VOCs play an important role in the ecology of aquatic environments.

Microbial degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantification using liquid chromatography-mass spectrometry.

The bacterial strain Sphingosinicella sp. B-9 was originally discovered to have the ability to degrade cyanobacterial cyclic peptides (microcystins), and has three hydrolytic enzymes (MlrA, MlrB, and MlrC). The purpose of this study was to examine in detail the degradation of glucagon/vasoactive intestinal polypeptide (VIP) family peptides by B-9, and to investigate the substrate specificity of B-9 proteases and the possibility of using a B-9 protease as a novel protease for peptide quantification by using a surrogate peptide and mass spectrometry (MS). The effective use of inhibitors revealed the following hydrolytic capability of B-9: One of the B-9 proteases (presumably MlrB) that was not inhibited by ethylenediaminetetraacetic acid (EDTA) cleaved bioactive peptides into medium-sized peptides with broad selectivity, similar to neutral endopeptidase, and another protease that was not inhibited by phenylmethylsulfonyl fluoride (PMSF) corresponded to MlrC and cleaved the resulting medium-sized peptides to smaller peptides or amino acids. The former property was desirable to obtain a suitable surrogate peptide, which was used successfully to quantify peptide using liquid chromatography (LC)-MS. Thus, the present study verified that one of the B-9 proteases has broad cleavage selectivity and cleavage sites, not seen in commercially available proteases, and is applicable to protein and peptide quantification using LC-MS.

Safety prediction of topically exposed biocides using permeability coefficients and the desquamation rate at the stratum corneum.

Advances in the synthesis and utilization of new chemical compounds have led to improvements in our daily lives. However, new chemicals may be both beneficial and toxic. Thus, exposure to these new compounds should be restricted in an attempt to limit their potential toxicities. We predicted the safety of three biocides (p-cresol, diazinon and resmethrin) by comparing their skin permeability coefficients and desquamation rate (the counter flux of permeability coefficient for chemical compounds induced by skin turnover) following skin exposure. In vitro skin permeation experiments revealed that the permeability coefficients of diazinon and resmethrin were smaller than the desquamation rate; therefore, these biocides could not permeate the skin, which resulted in very low skin concentrations of these compounds. On the other hand, the skin concentration of p-cresol was high because of its higher permeability coefficient than the desquamation rate. Furthermore, low in vitro cell viability was reported for skin exposed to p-cresol. These results in the present study indicate that the method described herein is useful for predicting the toxicities of chemicals following their topical exposure.

Volatile organic compounds derived from 2-keto-acid decarboxylase in Microcystis aeruginosa.

Volatile organic compounds (VOCs), 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol, were detected together with ß-cyclocitral from the cyanobacterium Microcystis aeruginosa NIES-843. These alcohols were optimally produced after 35 d of culture, during which nitrate nitrogen in the cultured broth became exhausted. Additionally, these alcohols were definitely produced using the 2-keto-acid decarboxylase (MaKDC) in Microcystis strains. These results suggested that these VOCs from Microcystis are significant for their lifecycle, because these compounds are not produced by any other genus of cyanobacteria. This is the first report of 2-keto-acid decarboxylase producing 3-methyl-1-butanol and 2-phenylethanol by an oxygenic photosynthetic microorganism.

Potencial de biodegradação de microcistinas por microrganismos / Biodegradation potential of microcystins by microorganisms

Para minimizar os problemas relacionados à ocorrência de cianobactéria em águas destinadas ao consumo humano há necessidade de se realizar estudos de alternativas técnicas de tratamento com destaque aos biofilmes com potencial de degradação de microcistinas (MC). O presente trabalho teve como objetivo avaliar o potencial de degradação de MC pela bactéria Sphingosinicella microcystinivorans B9, diferentes cepas de leveduras e bactérias probióticas. O teste foi efetuado com extrato de MC e diferentes quantidades de biovolume e densidade celular dos microrganismos. Os tratamentos foram mantidos a 27ºC com rotação de 100 rpm e as amostras para análise de MC e contagem dos microrganismos foram retiradas após 0 e 96 horas de contato. A bactéria B9 apresentou maior degradação de MC, chegando a 98% após 96 horas.

To minimize problems related to the occurrence of cyanobacteria in water for human consumption there is need to investigate alternative treatment techniques with emphasis on biofilms with the potential degradation of microcystins (MC). This study aimed to evaluate the potential degradation of MC by bacteria Sphingosinicella microcystinivorans B9, different strains of yeast and probiotic bacteria. The test was carried out with the extract obtained from strain Microcystis sp. In the tests biomass and cultures of microorganisms were used and the treatments were maintained at 27ºC with 100 rpm. Samples for analysis of MC and for counting the microorganisms were collected at 0 and 96 hours. The bacterium B9 presented the highest potential of degradation of MC reaching 98% after 96 hours.

Microbial degradation of physiologically active peptides by strain B-9.

The reaction of some physiologically active peptides with bacterial strain B-9 has been investigated. Bradykinin, ß-endorphin, and [Leu(5)]enkephalin were quickly degraded, with half-lives of <5 min. Somatostatin, substance P, and angiotensin I were degraded relatively smoothly, with half-lives of 10 min to 1 h, whereas oxytocin and insulin were slowly degraded, with half-lives of 1 and 4 days, respectively. Vasopressin was barely degraded, with a half-life of >7 days. Linearized vasopressin, prepared by the reductive cleavage of the disulfide bond followed by alkylation with iodoacetamide, was degraded significantly faster than intact vasopressin, with a half-life of 2.5 h. A loop formed by disulfide bond formation was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides in this study took place through cleavage of various peptide bonds, and the strain B-9 may bear similarities to the neutral endopeptidase in terms of its broad selectivity.

Analytical aspects of cyanobacterial volatile organic compounds for investigation of their production behavior.

In order to fully understand the role of volatile organic compounds (VOCs) under natural conditions, an adaptable analytical method was developed as the first step. beta-Ionone, beta-cyclocitral, 2-methyl-1-butanol and 3-methyl-1-butanol were simultaneously analyzed in addition to geosmin and 2-MIB using GC/MS with SPME. The slight modification of a known method allowed the simultaneous detection and quantification of these VOCs. The SIM of the 3-methyl-1-butanol was always accompanied by a shoulder peak, suggesting the presence of two compounds. In order to separate both compounds, the GC/MS conditions were optimized, and the additional peak was identified as 2-methyl-1-butanol by direct comparison of the authentic compound, indicating that the Microcystis strain always produces a mixture of 2-methyl-1-butanol and 3-methyl-1-butanol. Furthermore, it was found that 2-methyl-1-butanol and 3-methyl-1-butanol were predominant in the dissolved fractions. beta-Cyclocitral was easily oxidized to provide the oxidation product, 2,6,6-trimethylcyclohexene-1-carboxylic acid, which causes the blue color formation of cyanobacteria as a consequence of acid stress. The intact acid could be satisfactorily analyzed using the usual GC/MS without derivatization.

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1alpha,25-dihydroxyvitamin D3 synthesis in leptin-deficient mice.

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone-derived fibroblast growth factor 23 (FGF-23) as a mediator of the influence of leptin on renal 1alpha-hydroxylase mRNA expression in leptin-deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF-23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12-hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF-23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Administration of FGF-23 (5 microg i.p. at 12-hour intervals for 2 days) to ob/ob mice suppressed renal 1alpha-hydroxylase mRNA expression. The main site of FGF-23 mRNA expression was the bone, and leptin markedly increased the FGF-23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1alpha-hydroxylase and sodium-phosphate cotransporters (NaP(i)-IIa and NaP(i)-IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF-23 level and renal expression of 1alpha-hydroxylase mRNA were not influenced by administration of leptin to leptin receptor-deficient (db/db) mice. These results indicate that leptin directly stimulates FGF-23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)(2)D(3) synthesis in these mice is at least partly due to elevated bone production of FGF-23.

Blue color formation of cyanobacteria with beta-cyclocitral.

Volatile compounds, such as beta-cyclocitral, geosmin, and 2-methylisoborneol, from cyanobacteria showed a lytic activity against cyanobacteria. Particularly, beta-cyclocitral caused an interesting color change in the culture broth from green to blue during the lysis process. In the present study, the lytic behavior of various cyanobacteria with beta-cyclocitral was investigated, and a mechanism for the blue color formation was developed. beta-Cyclocitral lysed both the laboratory strains of any genera and bloom samples including many species of cyanobacteria, and caused the characteristic color change from green to blue. beta-Cyclocitral provided a characteristic behavior, such that the absorption maxima of chlorophyll-a and beta-carotene disappeared, but that of phycocyanin still remained after 12 h, which indicated that beta-cyclocitral decomposed chlorophyll-a and beta-carotene rapidly, so that the inherent colors from the tolerant water-soluble pigments became observable in the cultured broth. This phenomenon was confirmed by another experiment using Phormidium (NIES-611), which showed a pink color derived from phycoerythrin. beta-Cyclocitral was more easily oxidized when compared with similar aldehyde compounds, so that the pH of the solution quickly decreased to 4.5. An oxidation product of beta-cyclocitral in water solution was isolated and identified as 2,6,6-trimethylcyclohexene-1-carboxylic acid. This study provides support that beta-cyclocitral derived from cyanobacteria plays an important role in the lysis of cyanobacteria and participates in the blue color formation under natural conditions.

Ceramide biosynthesis in keratinocyte and its role in skin function.

The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1-CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.

Evaluation of synthetic sphingolipid analogs as ligands for peroxisome proliferator-activated receptors.

In this Letter, we assessed newly synthesized sphingolipid analogs as ligands for peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta or PPARgamma, using a dual-luciferase reporter system. We tested 640 sphingolipid analogs for ligand activity. As a result, seven types: A9, B9, C9, C50, F66, G66 and H66, were found to show agonistic activities for PPARs.

Microbial degradation of cyclic peptides produced by bacteria.

Bacterial strain, B-9, isolated from Lake Tsukui, Japan, and characterized as genus Sphingosinicella sp., possesses hydrolytic enzymes capable of degrading various toxic and non-toxic cyanobacterial cyclic peptides, such as microcystins, nodularin, microviridin, microcyclamide and aeruginopeptin. In this study, the degradation activities of the cell extract of B-9 against bacterial cyclic peptides, bacitracin, colistin, polymyxin, mikamycin, thiopeptin and WAP-8294A2, were investigated and the degradation products were analyzed using HPLC and liquid chromatography/ion trap tandem mass spectrometry (LC/ITMS). As a result of extensive experiments, it was confirmed that B-9 could also degrade these bacterial cyclic peptides by hydrolysis of their peptide or ester bonds, except for WAP-8294A2. These results indicated that the functions of the bacterium with its enzymes were further extended and offered the possibility of degrading other types of compounds.

Further investigation of microbial degradation of microcystin using the advanced Marfey method.

It is known that microcystin (MC) is subject to microbial degradation to provide three types of products, linearized MCLR (Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg), tetrapeptide Adda-Glu-Mdha-Ala, and Adda. They can be readily detected by the usual HPLC, because they commonly have an Adda moiety with a diene and an absorption maximum at 238 nm as the chromophore. However, no other degradation products without such a chromophore have been isolated to date. In this study, cell preparation of a bacterium B-9 that can degrade MC and detection of the degradation products were devised. First, we regulated the B-9 hydrolytic activity by washing with sodium chloride solution to obtain a desired cell preparation, which permitted an additional intermediate and the final products of MCLR to be obtained. Second, the resulting products could be firmly identified using the advanced Marfey method with the aid of log D. As a result of these experiments, the following degradation products were further identified: a tetrapeptide, Adda-Glu-Mdha-Ala, tripeptides Adda-Glu-Mdha, Glu-Mdha-Ala, and Arg-MeAsp-Leu, a dipeptide, Glu-Mdha, and amino acids Adda, Arg, and methylamine derived from Mdha. The present study expands the hydrolytic activity of the B-9 strain, which can hydrolyze not only cyanobacterial cyclic peptides but also MC to the intermediates and final products. The established characterization method composed of the advanced Marfey method and log D would be a standard technique for the structural characterization of a mixture of amino acids and peptides.