Anti-Inflammatory Activity of Lobaric Acid via Suppressing NF-κB/MAPK Pathways or NLRP3 Inflammasome Activation.

Lobaric acid (LA) is a constituent of the lichen Stereocaulon alpinum. LA has multiple biological activities, including antibacterial and antioxidant ones. The purpose of this study was to investigate the effect of LA and its mechanism on lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Macrophages were pretreated with different concentrations of LA (0.2 - 20 µM), followed by LPS stimulation. LA treatment of LPS stimulated macrophages decreased their nitric oxide production and the expression of cyclooxygenase-2 and prostaglandin E2. LA also significantly reduced the production of tumor necrosis factor-α and interleukin (IL)-6 by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). Additionally, LA inhibited the production of IL-1ß and IL-18, as well as caspase-1 maturation, by inhibition of NLRP3 inflammasome activation in LPS/ATP-stimulated cells. These results strongly suggest that LA could inhibit inflammation by downregulating NF-κB/MAPK pathways and NLRP3 inflammasome activation in activated macrophages. These results reveal a new therapeutic approach to modulate inflammatory diseases linked to deregulated inflammasome activities.

Leucothrix arctica sp. nov., isolated from Arctic seawater.

A Gram-stain-negative, non-motile, oxidase- and catalase-positive, rod-shaped bacterium was isolated from a coastal seawater sample from the Arctic Circle and designated strain IMCC9719T. On the basis of 16S rRNA gene sequence analysis, it was shown that strain IMCC9719T belonged to the genus Leucothrix and was closely related to the type strains of Leucothrix pacifica (97.6 % similarity) and Leucothrix mucor (95.1 %), while the strain shared <90.6 % sequence similarity with other bacterial species. The average nucleotide identity and genome-to-genome distance values between strain IMCC9719T and L. pacifica JCM 18388T were 71.7 and 16.9 %, respectively. The DNA G+C content of strain IMCC9719T was 43.5 mol%. Optimum growth of strain IMCC9719T was observed at 15 °C, at pH 7.5-8.5 and in the presence of 2.0-2.5 % (w/v) NaCl. The major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and C16 : 0. Cells of strain IMCC9719T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified polar lipid, an unidentified aminophospholipid and two unidentified phospholipids. The major respiratory quinone was ubiquinone-8 (Q-8). Based on the taxonomic data collected in this study, strain IMCC9719T represents a novel species of the genus Leucothrix, for which the name Leucothrix arctica sp. nov. is proposed. The type strain is IMCC9719T (=KACC 18010T=NBRC 110382T).

Planktotalea arctica sp. nov., isolated from Arctic seawater.

A Gram-staining-negative, non-motile, non-pigmented, rod-shaped bacterium was isolated from an Arctic coastal seawater sample and was designated strain IMCC9565T. Analysis of the 16S rRNA gene sequence of strain IMCC9565T revealed that the closest phylogenetic neighbours of the strain were members of the genus Planktotalea. Furthermore, the strain formed a robust clade with Planktotalea frisia SH6-1T, with which it shared 97.9 % 16S rRNA gene sequence similarity. Determination of genomic relatedness based on average nucleotide identity and genome-to-genome distance showed that strain IMCC9565T was distantly related to P. frisia, meaning the Arctic strain represents a novel species. Optimum growth of strain IMCC9565T was observed at 20 °C, pH 7.0 and in the presence of 2 % (w/v) NaCl. The major respiratory isoprenoid quinone was ubiquinone-10 (Q-10) and the major polar lipids consisted of phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified lipids. The principal fatty acids were C18 : 1ω7c and/or C18 : 1ω6c, C18 : 1ω7c 11-methyl and C16 : 0, and the DNA G+C content was 57.1 mol%. Based on these data, Planktotalea arctica sp. nov. is proposed to accommodate the bacterial isolate and the type strain is IMCC9565T (=KACC 18009T=NBRC 110393T).

Reclassification of Halomonas caseinilytica Wu et al. 2008 as a later synonym of Halomonas sinaiensis Romano et al. 2007, and emendation of the species description.

The taxonomic relationship between Halomonas sinaiensis DSM 18067(T) and Halomonas caseinilytica JCM 14802(T) has not been established, despite the high similarity (99.6 %) of their 16S rRNA gene sequences. To clarify their taxonomic positions, a polyphasic approach was applied to both type strains. Genomic relatedness analyses between H. sinaiensis DSM 18067(T) and H. caseinilytica JCM 14802(T) resulted in an average nucleotide identity of 99.5 % and an estimated DNA-DNA hybridization of 96.1 % by the genome-to-genome distance calculator, indicating that they belong to a single species. Phenotypic and chemotaxonomic characteristics showed no pronounced differences between the two type strains. Based on the results of this polyphasic study, it is proposed that H. caseinilytica JCM 14802(T) is a later heterotypic synonym of H. sinaiensis DSM 18067(T). An emended description for the species H. sinaiensis is given.

Ramalin-Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells.

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF-7 and MDA-MB-231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration-dependent manner. By upregulating Bax and downregulating Bcl-2, ramalin caused cytochrome c and apoptosis-inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase-8 and caspase-9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase-3 only in the MDA-MB-231 cells. Ramalin treatment also increased the levels of LC3-II and p62. Moreover, the inhibition of autophagy by 3-methyladenine or Atg5 siRNA significantly enhanced ramalin-induced apoptosis, which was accompanied by a decrease in Bcl-2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non-invasive or invasive breast cancer.

Ramalin Isolated from Ramalina Terebrata Attenuates Atopic Dermatitis-like Skin Lesions in Balb/c Mice and Cutaneous Immune Responses in Keratinocytes and Mast Cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that involves eczematous skin lesions with pruritic erythematous papules. In this study, we investigated the mitigating effects of ramalin, a component of the Antarctic lichen Ramalina terebrata against AD in vivo and in vitro. Oral administration of ramalin lessened scratching behaviors and significantly reduced both serum immunoglobulin E and IL-4 levels, and mRNA levels of IL-4 and IL-10 in AD-induced Balb/c mice. In vitro, treatment with ramalin produced significantly less inflammatory chemokines and cytokines, including TARC, MCP-1, RANTES, and IL-8 in TNF-α-stimulated HaCaT cells. In addition, ramalin inhibited the activation of nuclear factor-kappa B as well as the phosphorylation of mitogen-activated protein kinases (MAPK). Furthermore, ramalin treatment resulted in decreased production of ß-hexosaminidase and proinflammatory cytokines IL-4, IL-6, and TNF-α in 2,4 dinitrophenyl-human serum albumin-stimulated RBL-2H3 cells through blocking MAPK signaling pathways. The results suggest that ramalin modulates the production of immune mediators by inhibiting the nuclear factor-kappa B and MAPK signaling pathways. Taken together, ramalin effectively attenuated the development of AD and promoted the mitigating effects on Th2 cell-mediated immune responses and the production of inflammatory mediators in mast cells and keratinocytes. Thus, ramalin may be a potential therapeutic agent for AD. Copyright © 2016 John Wiley & Sons, Ltd.

Halocynthiibacter arcticus sp. nov., isolated from Arctic marine sediment.

A Gram-staining-negative, oxidase- and catalase-positive, non-motile, aerobic and rod-shaped bacterium producing white colonies, PAMC 20958T, was isolated from a marine sediment of the Arctic. PAMC 20958T grew at 10-27 °C (optimally at 21 °C), at pH 5.5-9.5 (optimally at pH 7.0-7.5) and in the presence of 0.5-7.5 % (w/v) (optimally at 2.0 %) NaCl. PAMC 20958T showed 97.5 % 16S rRNA gene sequence similarity with Halocynthiibacter namhaensis KCTC 32362T and formed a robust phylogenetic clade with this species. The average nucleotide identity value between strain PAMC 20958T and H. namhaensis KCTC 32362T was 79.7 % and the genome-to-genome distance was 13.0 % on average. The genomic DNA G+C content calculated from the genome sequence was 53.2 mol%. The major fatty acids were C18 : 1ω7c and/or C18 : 1ω6c. The major respiratory isoprenoid quinone was ubiquinone-10 (Q-10) and major polar lipids were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and two unidentified lipids. On the basis of phylogenetic analysis and genotypic and phenotypic data obtained in this study, it is concluded that strain PAMC 20958T ( = KCTC 42129T = JCM 30530T) represents the type strain of a novel species of the genus Halocynthiibacter, for which the name Halocynthiibacter arcticus sp. nov. is proposed.

Domibacillus tundrae sp. nov., isolated from active layer soil of tussock tundra in Alaska, and emended description of the genus Domibacillus.

A novel Gram-stain-positive, spore-forming, aerobic, motile and rod-shaped bacterium designated strain PAMC 80007T was isolated from an active layer soil sample of Council, Alaska. Optimal growth of strain PAMC 80007T was observed at 30 °C, pH 7.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain PAMC 80007T belonged to the genus Domibacillus. This strain was closely related to Domibacillus enclensis (98.3 %), Domibacillus robiginosus (98.3 %) and Domibacillus indicus (97.2 %). Genomic DNA G+C content was 43.5 mol% and genomic relatedness analyses based on the average nucleotide identity and the genome-to-genome distance showed that strain PAMC 80007T is clearly distinguished from the closely related species of the genus Domibacillus. The major fatty acids (>5 %) were iso-C15 : 0 (24.7 %), C16 : 1ω11c (16.8 %), anteiso-C15 : 0 (16.5 %), C16 : 0 (15.6 %) and anteiso-C17 : 0 (8.7 %). The major respiratory isoprenoid quinones were menaquinone-6 (MK-6) and menaquinone-7 (MK-7), and the polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, phospholipid and two unidentified lipids. meso-Diaminopimelic acid (type A1γ) was present in the cell-wall peptidoglycan, and the major whole-cell sugar was ribose with a minor quantity of glucose. Results from a polyphasic study suggested that strain PAMC 80007T represents a novel species of the genus Domibacillus for which the name Domibacillus tundrae sp. nov. is proposed. The type strain is PAMC 80007T ( = JCM 30371T = KCTC 33549T = DSM 29572T). An emended description of the genus Domibacillus is also provided.

Sediminicola arcticus sp. nov., a psychrophilic bacterium isolated from deep-sea sediment, and emended description of the genus Sediminicola.

A Gram-stain-negative, rod-shaped and non-motile strain, designated PAMC 27266(T), was isolated from deep-sea sediment of the Arctic Ocean. Analysis of the 16S rRNA gene sequence of strain PAMC 27266(T) showed closest affiliation with the genus Sediminicola . Phylogenetic analyses revealed that strain PAMC 27266(T) formed a robust clade with Sediminicola luteus CNI-3(T), with which it shared 98.9% 16S rRNA gene sequence similarity. Genomic relatedness analyses based on the average nucleotide identity and genome-to-genome distance showed that strain PAMC 27266(T) is clearly distinguished from S. luteus . Cells of strain PAMC 27266(T) grew optimally at 15 °C and pH 6.5-7.5 in the presence of 3.5% (w/v) sea salts. The major polar lipids were phosphatidylethanolamine, two unidentified aminophospholipids and two unidentified lipids. The only respiratory quinone was menaquinone-6. The major cellular fatty acids (>10%) were C16 : 1ω6c and/or C16 : 1ω7c and C15 : 0. The genomic DNA G+C content was 37.9 mol%. Based on the phylogenetic, genomic, chemotaxonomic and phenotypic data presented, we propose strain PAMC 27266(T) ( =KCCM 43038(T) =JCM 19894(T)) as the type strain of a novel species, with the name Sediminicola arcticus sp. nov.

Algibacter psychrophilus sp. nov., a psychrophilic bacterium isolated from marine sediment.

A Gram-stain-negative, aerobic, yellow-pigmented, flexirubin-negative, rod-shaped, non-motile and psychrophilic bacterial strain, PAMC 27237T, was isolated from marine sediment of the Ross Sea, Antarctica. Strain PAMC 27237T grew at 0-20 °C (optimally at 17 °C), at pH 5.0-9.5 (optimally at pH 7.0) and in the presence of 0-3.5 % (w/v) NaCl (optimally at 1.5-2.5 %). The major fatty acids (≥5 %) were iso-C17 : 0 3-OH, C17 : 0 2-OH, anteiso-C15 : 0, summed feature 3 (C16 : 1ω6c/C16 : 1ω7c), iso-C15 : 0 3-OH, anteiso-C17 : 1ω9c, anteiso-C15 : 1 A, iso-C16 : 0 3-OH and iso-C15 : 1 G. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, four unidentified lipids and a glycolipid. The major respiratory quinone was MK-6. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain PAMC 27237T belongs to the genus Algibacter, showing high similarities with the type strains of Algibacter agarivorans (97.2 %), Algibacter agarilyticus (97.0 %) and Algibacter mikhailovii (96.4 %). Average nucleotide identity values between strain PAMC 27237T and the type strains of A. agarivorans and A. agarilyticuswere 83.1 and 84.2 %, respectively, and mean genome-to-genome distances were 22.4-24.2 %, indicating that strain PAMC 27237T is clearly distinguished from the most closely related species of the genus Algibacter. The genomic DNA G+C content calculated from genome sequences was 33.5 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, strain PAMC 27237T is considered to represent a novel species of the genus Algibacter, for which the name Algibacter psychrophilus sp. nov. is proposed. The type strain is PAMC 27237T ( = KCTC 42130T = JCM 30370T).

Psychroserpens jangbogonensis sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment.

A Gram-staining-negative, yellow-pigmented, aerobic, rod-shaped and non-motile bacterium, PAMC 27130(T), was isolated from the marine sediment of the Ross Sea, Antarctica. The temperature, pH and NaCl tolerance ranges for growth were 4-20 °C, pH 6.0-9.0 and 0.5-5.0 % (w/v) NaCl, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PAMC 27130(T) belonged to the genus Psychroserpens and was closely related to Psychroserpens mesophilus, Psychroserpens damuponensis and Psychroserpens burtonensis with 97.2, 94.7 and 94.2 % sequence similarities, respectively. Genomic relatedness analyses based on average nucleotide identity and genome-to-genome distance showed that strain PAMC 27130(T) could be clearly distinguished from other species of the genus Psychroserpens . The genomic DNA G+C content was 32.7 mol%. The major fatty acids (>10 %) were C20 : 4ω6c (13.2 %), iso-C15 : 0 (12.3 %), iso-C15 : 1 G (11.7 %) and iso-C15 : 0 3-OH (10.0 %). The major respiratory isoprenoid quinone was menaquinone-6 (MK-6) and the polar lipids were phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, an unidentified aminophospholipid and three unidentified lipids. On the basis of genotypic and phenotypic data collected in this study, it is proposed that strain PAMC 27130(T) represents a novel species of the genus Psychroserpens, for which the name Psychroserpens jangbogonensis sp. nov. is proposed. The type strain is PAMC 27130(T) ( = KCTC 42128(T) = JCM 30228(T)).

Ramalin inhibits VCAM-1 expression and adhesion of monocyte to vascular smooth muscle cells through MAPK and PADI4-dependent NF-kB and AP-1 pathways.

Cell adhesion molecules play a critical role in inflammatory processes and atherosclerosis. In this study, we investigated the effect of ramalin, a chemical compound from the Antarctic lichen Ramalina terebrata, on vascular cell adhesion molecule-1 (VCAM-1) expression induced by TNF-α in vascular smooth muscular cells (VSMCs). Pretreatment of VSMCs with ramalin (0.1-10 µg/mL) concentration-dependently inhibited TNF-α-induced VCAM-1 expression. Additionally, ramalin inhibited THP-1 (human acute monocytic leukemia cell line) cell adhesion to TNF-α-stimulated VSMCs. Ramalin suppressed TNF-α-induced production of reactive oxygen species (ROS), PADI4 expression, and phosphorylation of p38, ERK, and JNK. Moreover, ramalin inhibited TNF-α-induced translocation of NF-κB and AP-1. Inhibition of PADI4 expression by small interfering RNA or the PADI4-specific inhibitor markedly attenuated TNF-α-induced activation of NF-κB and AP-1 and VCAM-1 expression in VSMCs. Our study provides insight into the mechanisms underlying ramalin activity and suggests that ramalin may be a potential therapeutic agent to modulate inflammation within atherosclerosis.

Lacinutrix jangbogonensis sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment and emended description of the genus Lacinutrix.

A Gram-negative, strictly aerobic, non-motile, rod-shaped and psychrophilic bacterial strain, PAMC 27137(T), was isolated from the marine sediment of the Ross Sea, Antarctica. Strain PAMC 27137(T) was observed to grow at 4-10 °C, at pH 6.5-7.5 and in the presence of 2.5-4.0 % (w/v) sea salts. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain PAMC 27137(T) belongs to the genus Lacinutrix showing the high similarities with Lacinutrix mariniflava JCM 13824(T) (97.6 %) and Lacinutrix algicola JCM 13825(T) (97.1 %). Genomic relatedness analyses based on the average nucleotide identity and the genome-to-genome distance showed that strain PAMC 27137(T) is clearly distinguished from the most closely related Lacinutrix species. The major fatty acids (>5 %) were identified as iso-C15:1 G (19.9 %), iso-C15:0 (19.3 %), iso-C17:0 3-OH (11.3 %), summed feature 9 (C16:0 10-methyl and/or iso-C17:1 ω9c as defined by MIDI, 9.1 %), iso-C15:0 3-OH (7.5 %), and anteiso-C15:1 A (5.8 %). The polar lipids were found to consist of phosphatidylethanolamine, an unidentified aminolipid, an unidentified aminophospholipid, and five unidentified phospholipids. The major respiratory quinone was identified as MK-6. The genomic DNA G+C content was determined to be 32.1 mol%. Based on the data from this polyphasic taxonomic study, strain PAMC 27137(T) is considered to represent a novel species of the genus Lacinutrix, for which the name Lacinutrix jangbogonensis sp. nov. is proposed. The type strain is PAMC 27137(T) (=KCTC 32573(T)=JCM 19883(T)).

Biodiversity and physiological characteristics of Antarctic and Arctic lichens-associated bacteria.

The diversity and physiological characteristics of culturable bacteria associated with lichens from different habitats of the Arctic and Antarctica were investigated. The 68 retrieved isolates could be grouped on the basis of their 16S rRNA gene sequences into 26 phylotypes affiliated with the phyla Actinobacteria, Bacteroidetes, Deinococcus-Thermus, and Firmicutes and with the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Isolates belonging to the Alphaproteobacteria were the most abundant, followed by those belonging to Actinobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, and Deinococcus-Thermus. Phylogenetic analysis showed that approximately 21 % of the total isolates represented a potentially novel species or genus (≤97 % sequence similarity). Strains belonging to the genera Sphingomonas, Frondihabitans, Hymenobacter, and Burkholderia were recovered from lichen samples from both geographic locations, implying common and important bacterial functions within lichens. Extracellular protease activities were detected in six isolates, affiliated with Burkholderia, Frondihabitans, Hymenobacter, Pseudomonas, and Rhodanobacter. Extracellular lipase activities were detected in 37 isolates of the genera Burkholderia, Deinococcus, Frondihabitans, Pseudomonas, Rhodanobacter, Sphingomonas, and Subtercola. This is the first report on the culturable bacterial diversity present within lichens from Arctic and Antarctica and the isolates described herein are valuable resources to decode the functional and ecological roles of bacteria within lichens. In addition, the low similarity (≤97 %) of the recovered isolates to known species and their production of cold-active enzymes together suggest that lichens are noteworthy sources of novel bacterial strains for use in biotechnological applications.

Polaribacter sejongensis sp. nov., isolated from Antarctic soil, and emended descriptions of the genus Polaribacter, Polaribacter butkevichii and Polaribacter irgensii.

A Gram-staining-negative, catalase- and oxidase-positive, non-motile bacterium, designated strain KOPRI 21160(T), was isolated from Antarctic soil. Based on 16S rRNA gene sequence analysis, strain KOPRI 21160(T) was found to belong to the genus Polaribacter. Sequence similarity between strain KOPRI 21160(T) and the type strains of species of the genus Polaribacter was 94.2-98.3 %. The nearest phylogenetic neighbours of strain KOPRI 21160(T) were Polaribacter butkevichii KCTC 12100(T) (98.3 % similarity) and Polaribacter irgensii KCTC 23136(T) (97.5 %). DNA-DNA relatedness was 50.6 %, between strain KOPRI 21160(T) and P. butkevichii KCTC 12100(T), and 45.2 % between strain KOPRI 21160(T) and P. irgensii KCTC 23136(T). Strain KOPRI 21160(T) grew at 4-37 °C and at pH 7.0-8.5. It could hydrolyse DNA, starch and Tweens 20, 40, 60 and 80. Menaquinone-6 (MK-6) was the only respiratory quinone, and iso-C15 : 0, iso-C15 : 0 3-OH and C15 : 1ω6c were the major cellular fatty acids. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and one unidentified lipid. The DNA G+C content was 30.0 mol%. Based on data from our polyphasic study, the organism is considered to represent a novel species of the genus Polaribacter, for which we propose the name Polaribacter sejongensis sp. nov. The type strain is KOPRI 21160(T) ( = KCTC 23670(T) = JCM 18092(T)). Emended descriptions of the genus Polaribacter, Polaribacter butkevichii Nedashkovskaya et al. 2005 and Polaribacter irgensii Gosink et al. 1998 are also proposed.

Optimization of cold-active chitinase production from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702.

In the present study, cultivation conditions and medium components were optimized using statistical design and analysis to enhance the production of Chi21702, a cold-active extracellular chitinase from the Antarctic bacterium Sanguibacter antarcticus KOPRI 21702. Identification of significant carbon sources and other key elements was performed using a statistical design technique. Chitin and glycerol were selected as main carbon sources, and the ratio of complex nitrogen sources to carbon sources was determined to be 0.5. Among 15 mineral components included in basal medium, NaCl, Fe(C6H5O7), and MgCl2 were found to have the most influence on Chi21702 production. The optimal parameters of temperature, initial pH, and dissolved oxygen level were found to be 25°C, 6.5, and above 30% of air saturation, respectively. The maximum Chi21702 activity obtained under the optimized conditions was 90 U/L. Through statistical optimization methods, a 7.5-fold increase in Chi21702 production was achieved over unoptimized conditions. Chi21702 showed relatively high activity, even at low temperatures close to 0°C. The information obtained in the present study could be applied to the production of cold-active endochitinase on a large scale, suitable for a process at low temperature in industry.

Actimicrobium antarcticum gen. nov., sp. nov., of the family Oxalobacteraceae, isolated from Antarctic coastal seawater.

A Gram-negative, non-motile, catalase- and oxidase- positive, strictly aerobic, and short rod-shaped bacterium that was designated strain KOPRI 25157(T) was isolated from coastal seawater sample in Antarctica. The temperature and pH ranges for growth on R2A agar were 10-20°C, and 5.0-10.0, respectively. Phylogenetic analyses of the 16S rRNA gene sequence of strain KOPRI 25157(T) showed it to belong to the family Oxalobacteraceae of the class Betaproteobacteria, and it formed a distinct clade from other recognized members of the family. DNA G + C content was 65.9 mol%. Major ubiquinone was Q-8. Predominant cellular fatty acids were C(16:1) ω7c/15 iso 2OH (56.4%) and C(16:1) (30.5%). Major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and unknown lipid. On the basis of these data, it is proposed that strain KOPRI 25157(T) is the representative of a novel genus, for which the name Actimicrobium gen. nov. is proposed in the family Oxalobacteraceae. The type strain for Actimicrobium antarcticum sp. nov. is KOPRI 25157(T) (=JCM 16673(T)=KCTC 23040(T)).

Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization.

An endochitinase was previously purified and the gene was cloned from the psychrophilic Antarctic bacterium, Sanguibacter antarcticus (KCTC 13143). In the present study, recombinant endochitinase, rChi21702, was expressed using a yeast expression system (Pichia pastoris) and codon optimization. The expressed rChi21702 was purified by Phenyl-Sepharose column chromatography. Optimal expression yielded 1-mg purified enzyme from 1-L bioreactor culture. When p-NP-(GlcNAc)(2) was used as a substrate, the specific activity of the enzyme was determined to be 20U/mg. In vitro assays and thin-layer chromatography demonstrated that the recombinant enzyme has endochitinase activity that produces diacetyl-chitobiose as a dominant end product when chitooligomers, colloidal chitin, and the chromogenic p-NP-(GlcNAc)(2) are used as substrates. Optimal activity for rChi21702 was observed at 37 degrees C and a pH of 7.6. Interestingly, rChi21702 exhibited 63% of optimal activity at 10 degrees C and 44% activity at 0 degrees C. Taken together, the results indicate that rChi21702 has psychrotolerant endochitinase activity even after recombinant expression in yeast cells.

Human dermal fibroblast proliferation activity of usimine-C from Antarctic lichen Ramalina terebrata.

Type I collagen is the major structural protein in dermis and its presence is used to monitor skin cell proliferation and aging. Recently, novel usimine compounds have been found in the Antarctic lichen Ramalina terebrata. In the present study, usimine-C induced cell proliferation of human dermal fibroblast, CCD-986SK, up to 1.6-fold after treating with 90 microg/ml for 48 h. Type I procollagen synthesis was significantly increased 1.3-fold, 3-fold, and 5-fold after treating with 0.14, 0.72, and 3.6 microg usimine-C/ml for 24 h, respectively, whereas no significant increase in type I procollagen was observed after treating with usimine-A or -B. Usimines are usnic acid derivatives. Considering that the difference among the derivatives is a side chain, the proliferation activity may be related to this side chain, triggering an internal signal for type I procollagen expression. Further studies still remain to clarify the signaling pathways for the type I procollagen induction, which is activated by usimine-C.

Antibacterial activities of Ramalin, usnic acid and its three derivatives isolated from the Antarctic lichen Ramalina terebrata.

The development of new antibacterial compounds is an urgent issue to meet the evolution of resistivity of pathogenic bacteria against the available drugs. The objective of this study was to investigate the antibacterial compounds from the Antarctic lichen species Ramalina terebrata. A total of five compounds, usnic acid, usimine A, usimine B, usimine C, and ramalin, were isolated by bioactivity guided-fractionation of the methanol extract of R. terebrata after several chromatographic procedures. The qualitative antibacterial activities of the crude extract and isolated compounds were determined by the disk diffusion method while the minimum inhibitory concentration (MIC) determination assay gave the quantitative strength of the test samples. All the test samples showed antibacterial activity against Bacillus subtilis. The crude extract and usnic acid showed antibacterial activity against Staphylococcus aureus. The MIC values of the isolated compounds against B. subtilis were in the range of 1 to 26 microg/mL. These observed experimental data showed the strong antibacterial potential of these compounds against B. subtilis.