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The effects of diatom abundance and biogenic silica (BSi) deposition on the population growth of tintinnid ciliates were investigated using incubations of sub-surface waters (10 m depth) of Suruga Bay, Japan in April and November 2018. Seawater for incubation was pre-filtered using a 200-µm mesh to remove mesozooplankton grazers. Initial concentrations of dissolved inorganic nitrogen were similar in both seasons, but silicic acid and phosphate were higher in April than in November. This allowed us to observe the growth of planktonic communities under varied silicic acid availability and explore the effects of BSi accumulation and food availability on the growth of tintinnids. Phytoplankton blooms dominated by diatoms formed in both, April and November incubations, but their biomass was higher in April than in November, demonstrating the effects of nutrients, particularly silicic acid, on diatom growth. The BSi concentration increased in both incubations but was higher in April than in November, confirming the effects of diatom abundance and size on BSi deposition. Tintinnid biomass also increased in both incubations but was higher in April than in November, showing that the biomass of tintinnid was affected by the BSi concentration. Species composition of diatoms affected the species composition of tintinnids by providing selected material for the construction of their lorica. Moreover, the results showed that the food supply mainly provided by pico-nanoflagellates via microbial loop and detritus may limit tintinnids’ population growth in natural planktonic ecosystems and this demonstrates tintinnids’ role as top predators in the microbial loop

Porphyromonas gingivalis and Tannerella forsythia have been thought to be associated with periodontitis; however comprehensive histopathological localization of bacteria in affected human periodontal tissues is not well documented. In the present study, we examined formalin-fixed paraffin-embedded gingival and subgingival granulation tissues from 71 patients with chronic periodontitis and 11 patients with aggressive periodontitis, using immunohistochemistry with novel monoclonal antibodies specific to P. gingivalis or T. forsythia, together with quantitative real-time polymerase chain reaction for each bacterial DNA. Immunohistochemisty revealed both bacterial species extracellularly, as aggregates or within bacterial plaque, and intracellularly in stromal inflammatory cells, squamous epithelium, and capillary endothelium of granulation tissue. Combined analysis with the results from polymerase chain reaction suggested that localization and density of T. forsythia is closely associated with those of P. gingivalis, and that bacterial density is a factor responsible for the cell-invasiveness and tissue-invasiveness of these periodontal bacteria. Detection of these bacteria in the capillary endothelium in some samples suggested possible bacterial translocation into the systemic circulation from inflamed gingival and subgingival granulation tissues. Immunohistochemistry with the novel antibodies showed high specificity and sensitivity, and can be used to locate these periodontal bacteria in routinely-used formalin-fixed paraffin-embedded human tissue sections from systemic locations.

Ocean surface acidification due to increasing atmospheric CO2 concentration is currently attracting much attention. Coccolithophores distribute widely across the world's oceans and represent a carbon sink containing about 100 million tonnes of carbon. For this reason, there is concern about dissolution of their shells, which are made of calcium carbonate, due to decreasing pH. In this study, intracellular calcification, photosynthesis, and mass transport through biomembranes of Emiliania huxleyi were modelled numerically for understanding biological response in calcifying organisms. Unknown parameters were optimised by a generic algorithm to match existing experimental results. The model showed that the production of calcium carbonate rather than its dissolution is promoted under an acidified environment. Calcite remains at saturation levels in a coccolith even when it is below saturation levels in the external seawater. Furthermore, a coccolith can dissolve even in water where calcite saturation exceeds 1, because the saturation may be below the threshold level locally around the cell membrane. The present model also showed that the different calcification rates of E. huxleyi with respect to rising CO2 concentrations reported in the literature are due to differences in experimental conditions; in particular, how the CO2 concentration is matched. Lastly, the model was able to reproduce differences in calcification rates among coccolithophore species. The above biochemical-kinetic model was then incorporated into an ecosystem model, and the behaviour of coccolithophores in the ecosystem and the influence of increases in CO2 concentration on water quality were simulated and validated by comparison with existing experimental results. The model also suggests that increased CO2 concentration could lead to an increase in the biomass ratio of coccolithophores to diatoms at high CO2 concentrations, particularly in oligotrophic environments, and to a consequent decrease in pH due to calcium dissolution.

This study explored the physiological responses of the coral Pocillopora damicornis to high nitrate concentrations and thermal stresses. The expression of heat shock proteins Hsp60 and Hsp32, Symbiodiniaceae density, Chl a concentration, Fv/Fm, H2O2 scavenging, and caspase 3 activity varied during 60 h incubations at 28 °C or 32 °C, ambient or high nitrate (~10 μM) concentrations, and their combinations. In combined stresses, corals showed a rapid and high oxidation level negatively affecting the Symbiodiniaceae density and Chl a concentration at 12 h, followed by caspase 3 and Hsps upregulations that induced apoptosis, bleaching and tissue detachment. Corals under thermal stress showed the highest oxidation and upregulation of Hsps and caspase 3 resulting in coral discoloration. High nitrate treatment alone did not seriously affect the coral function. Results showed that combined stress treatment severely affected coral physiology and, judging from the condition of detached tissues, these corals might have lower chances to recover.

Urea is an unstable and intermediate organic nitrogenous compound present in coastal environments and is derived from the excretion of some aquatic organisms and wastewater discharges. Urea plays an important role in the nitrogen cycle, where it is utilized by algae, including diatoms. However, there are very limited relevant data on the production, consumption, and degradation of urea because of the lack of appropriate measurement techniques. The conventional method is based on the formation of a colored product when urea reacts with diacetyl monoxime in a sulfuric acid solution. We examined the optimal conditions for the formation of the colored product; specifically, we evaluated different temperatures (22–80 °C), reaction times, mixing ratios of color reagents, and sample storage times. Application of the single mixed color-developing reagent (COLDER) at 70 °C resulted in the optimal formation of the colored product within a short reaction time of 60 min. This method was then used to measure dissolved urea in different coastal environments. The concentrations detected were as follows: 0.65–0.72, 0.49–0.58, and 1.09–2.28 µM urea-N at coral reef, seagrass, and mangrove sites, respectively. Our results showed high precision (SD = 0.02, CV = 1.2%), a low detection limit (0.03 µM urea-N), and a high recovery rate (94–99%). In summary, this high-temperature procedure for urea measurements should be valuable for obtaining high-precision data that can further the understanding of urea dynamics and its role in coastal ecosystems.

The feeding strategies of Montipora digitata and Porites lutea, two dominant corals in the Okinawan reefs, were investigated. The focus was on pico- and nanoplankton feeding efficiencies, using 6-h incubations. Although healthy M. digitata consumed from 72% to 87% more pico-nanoplankton cells than P. lutea, feeding rates of bleached corals of both species were similarly low at heat stress (33 °C). Heterotrophic carbon acquisition with respect to dark respiration varied from 3% to 65% in M. digitata and from 7% to 68% in P. lutea. A decrease in the feeding efficiency of bleached M. digitata under heat stress shows its vulnerability to water heating events. Feeding rates of P. lutea were low under all conditions and treatments; therefore, this species is less vulnerable to heat stress due to the strategy of meeting metabolic costs by using translocated organic matter from endoliths and selecting pico-nanoplankton cells with a high C/N ratio.

Elevated seawater temperatures in the summer months of 2010 were associated with widespread coral mortality in Thailand. A large number of corals at Mu Ko Surin died following the bleaching event. Understanding of the recruitment of corals would improve our ability to predict the potential for coral recovery from the impacts of bleaching events, as well as the interpretation of spatio-temporal variability in coral community structure. This study aims to examine the composition, abundance and growth rate of juvenile corals and the potential of reef recovery at Mu Ko Surin in order to help to understand how reefs react to major disturbances. We found that the densities of coral recruits varied among years and study sites. In the year 2011, coral recruitments ranged between 0.18 ± 0.02 to 1.67 ± 0.07 recruits per m2 for 10 study sites. While in 2012, the monitoring revealed a range between 0.96 ± 0.16 and 2.19 ± 0.21 recruits per m2 from 5 study sites. Fungia, Acropora, Porites and Favites were the dominant groups of coral recruits. In terms substrate forms, they were significant differences between sampling years but the preferential dominant substrate forms did not differ. The Acropora recruits at Ko Torinla showed normal distributions of size class during the two periods. Their ranges in 2011 and 2012 were 4–30 and 13–54 mm, respectively. Six species of Acropora recruits, i.e. Acropora intermedia, A. nasuta, A. cerealis, A. subulata, A. muricata and A. latistella were found. They showed diverse growth rates due to the spatial distribution of 2.11 ± 0.59 to 7.47 ± 1.37 cm per year. This study provides useful data in terms of coral recruitment and recovery from degradation and disturbance, especially from temperature changes induced by coral bleaching. The findings suggest that there is the possibility for coral recovery around Mu Ko Surin following the 2010 bleaching event.

Corals evolved by establishing symbiotic relationships with various microorganisms (the zooxanthellae, filamentous algae, cyanobacteria, bacteria, archaea, fungi and viruses), forming the ‘coral holobiont'. Among them, the endolithic community is the least studied. Its main function was considered to be translocation of photo-assimilates to the coral host, particularly during bleaching. Here, we hypothesize that (i) endolithic algae may show similar primary production rates in healthy or bleached corals by changing their pigment ratios, and therefore that similar production and translocation of organic matter may occur at both conditions and (ii) diazotrophs are components of the endolithic community; therefore, N 2 fixation and translocation of organic nitrogen may occur. We tested these hypotheses in incubation of Porites lutea with 13 C and 15 N tracers to measure primary production and N 2 fixation in coral tissues and endoliths. Assimilation of the 13 C atom (%) was observed in healthy and bleached corals when the tracer was injected in the endolithic band, showing translocation in both conditions. N 2 fixation was found in coral tissues and endolithic communities with translocation of organic nitrogen. Thus, the endolithic community plays an important role in supporting the C and N metabolism of the holobiont, which may be crucial under changing environmental conditions.

Three novel Vibrio phages were isolated from seawater in Okinawa. The Vibrio phage RYC infected Vibrio coralliilyticus SWA 07, while Vibrio phages CKB-S1 and CKB-S2 infected the coral pathogen V. coralliilyticus P1 (LMG 23696). The Vibrio phages CKB-S1 and CKB-S2 displayed head-tail structures whereas the Vibrio phage RYC showed a tailless non-enveloped capsid. All these Vibrio phages contained linear and double-stranded DNA. The whole genome sequencing revealed that Vibrio phage RYC has a larger genome size compared to Vibrio phages CKB-S1 and CKB-S2, and six tRNAs genes were found only in Vibrio phage RYC. Genome-wide comparison showed that Vibrio phage CKB-S1 was closely related, but was not identical, to Vibrio parahaemolyticus phages VP16T and VP16C. Meanwhile, the Vibrio phages RYC and CKB-S2 did not show high genome-wide similarity to any phages. These results suggest that the Vibrio phages CKB-S1, CKB-S2 and RYC are novel phages, which need further exploration, especially for their potential applications in phage therapy.

A new polyacetylene carboxylic acid named montiporic acid D (1) was isolated along with a known polyacetylene alcohol, (Z)-13,15-hexadecadien-2,4-diyn-1-ol (2) from scleractinian coral Montipora digitata. The structures of compounds were determined by analyses of NMR and MS spectra.

Demersal zooplankton (DZ) appear in the water column at night, and are highly abundant in mangrove, seagrass, and coral reef habitats; however, few studies have discussed their role in aquatic food webs, considering different consumers and their preferences on different DZ’ size classes. This study elucidates the role of DZ as a food source for higher trophic levels in an estuarine area, particularly with respect to the food preference and size selection of their consumers. The study was conducted in the mangrove forest of Fukido Estuary and an adjacent reef lagoon (with seagrass-dominated and seagrass-coral mixture areas) on Ishigaki Island, Japan. The abundance of demersal zooplankton was 4.0, 5.4, and 11.3×10 4 ind.m -2 for seagrass, mangrove, and seagrass-coral mixture habitats, respectively. The lowest DZ biomass was recorded in mangroves and mainly dominated by smaller organisms, because their consumers in this habitat prefer large-sized prey. The δ 13 C and δ 15 N signatures showed that, in mangroves, demersal zooplankton constituted a higher proportion of the diet of fishes than in lagoon habitats; however, demersal zooplankton did not have a significant role in the diet of fishes and macroinvertebrates in the lagoon. Consistency among biomass, stomach contents, and the proportions of DZ of all size classes in the diet of mangrove fishes indicated that DZ serve as a major food source. In contrast, fishes in lagoon habitats consumed more crabs, shrimps and mollusks than DZ. In conclusion, our analytical approach allowed us to demonstrate that DZ of different body sizes serve as food sources for different consumers in different habitats of the estuarine ecosystem.

Increasing seawater temperatures and CO2 levels associated with climate change affect the shallow marine ecosystem function. In this study, the effects of elevated seawater temperature and partial pressure of CO2 (pCO2) on subtropical sediment systems of mangrove, seagrass, and coral reef lagoon habitats of Okinawa, Japan, were examined. Sediment and seawater from each habitat were exposed to four pCO2-temperature treatments, including ambient pCO2- ambient temperature, ambient pCO2-high temperature (ambient temperature + 4°C), high pCO2 (936 ppm)-ambient temperature, and high pCO2-high temperature. Parameters including primary production, nutrient flux, pigment content, photosynthetic community composition, and bacterial abundance were examined. Neither high temperature nor high pCO2 alone impacted mangrove and seagrass sediment primary production significantly (Tukey's test, P > 0.05). However, the combined stress significantly (Tukey's test, P < 0.01) increased primary production in these two habitats. In sediments from the coral reef lagoon, single and combined stress treatments induced a shift from heterotrophy to autotrophy. Significant increases in net primary production (Tukey's test, P < 0.01), and gross primary production (Tukey's test, P < 0.05) under the combined stress suggested that benthic microalgae in mangrove and seagrass sediments were more responsive to high temperature and pCO2 conditions than those in coral reef lagoon sediments. Additionally, combined stress significantly increased the sediment chlorophyll a content (Tukey's test, P < 0.05) in all habitats. These increases were associated with increased net primary production, indicating that combined stress stimulates primary production activity by the photosynthetic benthic microalgae in all habitats. Diatom activity increased, as silicate uptake increased in all habitats. Microbial abundance significantly increased under the combined stress treatment (Tukey's test, P < 0.01), but did not exceed autotrophic activity. Respiration did not change significantly in any of the three habitats (Tukey's test, P > 0.05) under the combined stress, suggesting that heterotrophic processes were less affected by the combined stress than autotrophic processes. In summary, mangrove and seagrass sediments minimize the negative impacts of elevated temperature and pCO2 via increased primary production and carbon storage. Lagoonal sediments also act as a carbon sink under temperature and ocean acidificationstress.

We describe a strategy to identify stress-responsive genes in corals by means of high-coverage cDNA expression profiling (HiCEP). In this study, the effects of thermal and chemical (DCMU and TBT-Cl) stresses on Acropora tenuis juvenile polyps were examined, and 19 genes were identified as stress-responsive genes. Of these, seven had sequences significantly similar to sequences of known proteins. We quantified the expression levels of these seven using real-time PCR. In corals, the genes encoding heat shock protein 90, calreticulin, methionine adenosyltransferase, and aquaglyceroporin showed differential stress-related expression patterns. In the endosymbiotic algae associated with the corals, genes coding for DnaJ-like protein and chloroplast light-harvesting protein were identified as stress-responsive genes. The only gene showing increased expression in response to all three stresses (thermal and both chemical stresses) was the coral gene coding for the oxidative stress-responsive protein, suggesting that oxidative stress generally occurs in each stress-exposed polyp.

Other than coral bleaching, few coral diseases or diseases of other reef organisms have been reported from Japan. This is the first report of lesions similar to Porites ulcerative white spots (PUWS), brown band disease (BrB), pigmentation response (PR), and crustose coralline white syndrome (CCWS) for this region. To assess the health status and disease prevalence, qualitative and quantitative surveys (3 belt transects of 100 m² each on each reef) were performed in March and September 2010 on 2 reefs of the Ginowan-Ooyama reef complex off Okinawa, and 2 protected reefs off Zamani Island, in the Kerama Islands 40 km west of Okinawa. Overall, mean (±SD) disease prevalence was higher in Ginowan-Ooyama (9.7 ± 7.9%) compared to Zamami (3.6 ± 4.6%). Porites lutea was most affected by PUWS at Ooyama (23.1 ± 10.4 vs. 4.5 ± 5.2%). White syndrome (WS) mostly affected Acropora cytherea (12. 5 ± 18.0%) in Zamami and Oxipora lacera (10.2 ± 10%) in Ooyama. Growth anomalies (GA) and BrB were only observed on A. cytherea (8.3 ± 6.2%) and A. nobilis (0.8%) at Zamami. Black band disease affected Pachyseris speciosa (6.0 ± 4.6%) in Ooyama only. Pigmentation responses (PR) were common in massive Porites in both localities (2.6 ± 1.9 and 5.6 ± 2.3% respectively). Crustose coralline white syndrome (CCWS) was observed in both localities. These results significantly expand the geographic distribution of PUWS, BrB, PR and CCWS in the Indo-Pacific, indicating that the northernmost coral reefs in the western Pacific are susceptible to a larger number of coral diseases than previously thought.

Methyl viologen (MV) causes severe oxidative stress by generating superoxide in the photosystem. The marine Chlamydomonas strain W80 is highly tolerant to MV (inhibitory concentration 50% [IC₅₀]=110 μM), and another marine Chlamydomonas strain HS5 shows also relatively a high tolerance (IC₅₀=12 μM). These two marine strains and a freshwater Chlamydomonas reinhardtii, which is highly sensitive to MV (IC₅₀=0.03 μM), were compared with respect to their reactive oxygen species (ROS) eliminating enzymes (superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase), intracellular free amino acids, and antioxidant activities of the cell extracts. The marked difference between the marine Chlamydomonas strains and C. reinhardtii is the much higher (more than 5 fold) ascorbate peroxidase (APX) activity in the marine strains. The marine strains also kept the high APX activities (more than 100% of non-stressed condition) under the MV stressed condition, while the APX activity in C. reinhardtii was significantly decreased (36% of non-stressed condition) under the stressed condition, indicating that APX activity potentially contributes to the oxidative stress tolerance in Chlamydomonas. In addition, the levels of intracellular free proline, which is supposed to ameliorate oxidative stress, were several tens of times higher in the marine Chlamydomonas strains than in C. reinhardtii.

All corals have a common structure: two tissue layers enclose a lumen, which forms the gastric cavity. Few studies have described the processes occurring inside the gastric cavity and its chemical and biological characteristics. Here, we show that the coral gastric cavity has distinct chemical characteristics with respect to dissolved O2, pH, alkalinity, and nutrients (vitamin B12, nitrate, nitrite, ammonium, and phosphate) and also harbors a distinct bacterial community. From these results, the gastric cavity can be described as a semi-closed sub-environment within the coral. Dissolved O2 shows very low constant concentrations in the deepest parts of the cavity, creating a compartmentalized, anoxic environment. The pH is lower in the cavity than in the surrounding water and, like alkalinity, shows day/night variations different from those of the surrounding water. Nutrient concentrations in the cavity are greater than the concentrations found in reef waters, especially for phosphate and vitamin B12. The source of these nutrients may be internal production by symbiotic bacteria and/or the remineralization of organic matter ingested or produced by the corals. The importance of the bacteria inhabiting the gastric cavity is supported by the finding of a high bacterial abundance and a specific bacterial community with affiliation to bacteria found in other corals and in the guts of other organisms. The findings presented here open a new area of research that may help us to understand the processes that maintain coral health.

This ten-year study examined the morphological, physiological, and ecological characteristics of coral growth anomalies on Acropora cytherea on Amuro Island, Okinawa, Japan. The objectives of the study were to assess whether the growth anomalies, identified as diffuse disruptions on the skeleton: (i) were more prevalent on large colonies than on small colonies, (ii) were more common near the center of the colonies than peripherally, (iii) affected colony growth and mortality, and (iv) affected coral-colony fecundity and photosynthetic capacity. We hypothesized that the growth anomalies were signs of the onset of aging. The growth anomalies were more prevalent on colonies >2 m diameter, and were concentrated near the central (older) portions of the colonies. The growth anomalies were also associated with reduced productivity and dysfunctional gametogenesis. Still, the growth anomalies did not appear to affect colony survival. The contact experiments showed that the growth anomalies were not contagious, and were most likely a sign of aging that was exacerbated by thermal stress.

Synechococcus is widely distributed in the world's ocean surfaces, and is often found in sediment traps. However, its distribution and ecological importance have not been well studied in meso- and bathypelagic waters. We measured Synechococcus abundance in the Suruga Bay (central Japan) and the subtropical NW Pacific. Synechococcus abundance at depths of 200 m and below var- ied from 2.4 to 190 cells ml -1 , but was in proportion to the surface abundance, suggesting transport of epipelagic populations to greater depths. Surprisingly, Synechococcus was evenly distributed from 200 m down to 1420 m (Suruga Bay) or to 2000 m (subtropical NW Pacific), regardless of season. The contribution of deep Synechococcus to the total population was highest in spring in the Suruga Bay (36 to 77%), and lowest in summer in the Suruga Bay (1 to 9%) and in the subtropical NW Pacific (4 and 10%). These results suggest effective transport of Synechococcus cells down the water column during productive seasons by attachment to large particles and limited transport under oligotrophic and stratified conditions. Deep Synechococcus abundance decreased from fall to winter in the Suruga Bay, though in filtered deep seawater it did not significantly decrease for 30 d in the dark, and it increased in a light/dark cycle. Our investigations show that the standing stock of Synechococcus has been significantly underestimated in previous studies of epipelagic waters conducted during produc- tive seasons and that Synechococcus seems to be grazed and to contribute to biogeochemical cycles in the dark ocean.

Various stresses induce the formation of reactive oxygen species (ROS) in biological cells. In addition to stress-induced ROS, we studied the photochemical formation of hydroxyl radicals (˙OH), the most potent ROS, in coral tissues using phosphate buffer-extracted solutions and a simulated sunlight irradiation system. ˙OH formation was seen in extracts of both coral host and endosymbiont zooxanthellae. This study is the first to report quantitative measurements of ˙OH photoformation in coral tissue extracts. Our results indicated that whether or not coral bleaching occurred, coral tissues and symbiotic zooxanthellae have the potential to photochemically produce ˙OH under sunlight. However, no significant difference was found in the protein content-normalized formation rates of ˙OH between corals incubated under different temperatures and irradiance conditions. ˙OH formation rates were reduced by 40% by reducing the UV radiation in the illumination. It was indicated that UV radiation strongly affected ˙OH formation in coral tissue and zooxanthellae, in addition to its formation through photoinhibition processes.

Production (abundance and biomass) and net calcification rates of the coccolithophorid Pleurochrysis carterae under different partial pressures of CO2 (pCO2) were examined using short (15, 24 and 39 h), long (7 d) and dark (7 d) incubation experiments. Short incubations were conducted at ambient, 500 and 820 ppm pCO2 levels in natural seawater that was enriched with nutrients and inoculated with P. carterae. Long incubations were conducted at ambient and 1200 ppm pCO2 levels in natural seawater (0.2 µm filtered as well as unfiltered) that was enriched with nutrients and inoc- ulated with P. carterae. Dark incubations were conducted at ambient and 1200 ppm pCO2 in unfil- tered seawater that was inoculated with P. carterae. The abundance and biomass of cocco- lithophorids increased with pCO2 and time. The abundance and biomass of most noncalcifying phytoplankton also increased, and were hardly affected by CO2 inputs. Net calcification rates were negative in short incubations during the pre-bloom phase regardless of pCO2 levels, indicating dis- solution of calcium carbonate. Further, the negative values of net calcification in short incubations became less negative with time. Net calcification rates were positive in long incubations during blooms regardless of pCO2 level, and the rate of calcification increased with pCO2. Our results show that P. carterae may adapt to increased (~1200 ppm) pCO2 level with time, and such increase has little effect on the ecology of noncalcifying groups and hence in ecosystem dynamics. In dark incuba- tions, net calcification rates were negative, with the magnitude being dependent on pCO2 levels.

Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean. Rapidly expanding benthic miniblooms, frequently dominated by a single cyanobacterial taxon, were identified by microscopy and molecular tools. In addition, nitrogenase activity by these blooms was measured in situ. Dinitrogen fixation and its contribution to mat primary production were calculated using (15)N(2) and (13)C methods. Dinitrogen-fixing cyanobacteria from mats in La Reunion and Sesoko showed few differences in taxonomic composition. Anabaena sp. among heterocystous and Hydrocoleum majus and Symploca hydnoides among nonheterocystous cyanobacteria occurred in microbial mats of both sites. Oscillatoria bonnemaisonii and Leptolyngbya spp. occurred only in La Reunion, whereas Hydrocoleum coccineum dominated in Sesoko. Other mats dominated by Hydrocoleum lyngbyaceum, Phormidium laysanense, and Trichocoleus tenerrimus occurred at lower frequencies. The 24-h nitrogenase activity, as measured by acetylene reduction, varied between 11 and 324 nmoles C(2)H(2) reduced microg(-1) Chl a. The highest values were achieved by heterocystous Anabaena sp. performed mostly during the day. Highest values for nonheterocystous cyanobacteria were achieved by H. coccineum mostly during the night. Daily nitrogen fixation varied from nine (Leptolyngbya) to 238 nmoles N(2) microg(-1) Chl day(-1) (H. coccineum). Primary production rates ranged from 1,321 (S. hydnoides) to 9,933 nmoles C microg(-1) Chl day(-1) (H. coccineum). Dinitrogen fixation satisfied between 5% and 21% of the nitrogen required for primary production.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. Propionibacterium acnes is the only microorganism so far isolated from sarcoid lesions. To examine whether P. acnes isolates from sarcoid tissues differ from those obtained from non-sarcoid tissues, we studied cell invasiveness, serotype, and polymorphisms of the P. acnes trigger factor protein and the two invasion-associated proteins (named PAmce and PAp60) in 35 P. acnes isolates from sarcoid lymph nodes and 127 isolates from non-sarcoid tissues. Most of the serotype I isolates (79/112; 71%), but none of the serotype II isolates (0/50) were cell-invasive. Two prominent types of trigger factors, one with and one without a 15 amino acid-residue deletion, corresponded to serotype II and serotype I, respectively. Non-invasive isolates had genomic mutations that caused more than one amino acid change in either the PAmce or PAp60 gene, with four exceptional isolates. P. acnes was finally classified into nine isotypes, and isolates obtained from sarcoid and non-sarcoid tissue did not differ. Although the finding did not link P. acnes to sarcoidosis, the present study clarified the cell invasiveness of P. acnes and the close correlation of cell invasiveness to the serotype and genotype of the two invasion-associated P. acnes genes.

Historically, hermatypic corals are defined as a symbiotic system composed of an animal host, corals belonging to the taxa Cnidarian, and a photosynthetic organism, the dinoflagellate Symbiodinium spp., also known as zooxanthellae. The high gross productivity and stability of coral reefs have been explained by the efficiency of the coral-algal symbiotic system in using the low nutrient concentrations found in the surrounding water and their rapid recycling in the water. Although several studies have reported the presence of bacteria closely associated with corals, the mechanisms of the relationships among them, the host and the zooxanthellae, remain to be shown. In this study, evidence for the importance of coelenteric bacteria in corals as a component of the coral symbiotic complex was shown by using a new approach. Vitamin B12, which is produced only by prokaryotes, was chosen as a chemical tool to clarify the symbiotic relationships among bacteria, coral, and zooxanthellae, which require vitamin B12. High vitamin B12 concentrations (up to 700pmol l-1 compared with max. 20pmol l-1 in the surrounding water) and high bacteria abundances (100 times higher than surrounding water) were found in the coelenteron of live corals using a new sampling method. The results led to the hypothesis that prokaryotes are the drivers of internal processes, such as vitamin B12 production, occurring directly inside the coral, forming a semi-closed system. Furthermore, most maintenance of the symbiotic complex is due to internal processes rather than the supply from outside the coral.

Helicobacter pylori has been considered to be non-invasive and to rarely infiltrate the gastric mucosa, even though there is an active Th1 immune response in the lamina propria of the H. pylori-infected stomach. To elucidate whether H. pylori invades the lamina propria and translocates to the gastric lymph nodes, we examined H. pylori in formalin-fixed and paraffin-embedded tissue sections of stomach and gastric lymph nodes obtained from 51 cancer patients using real-time PCR and immunohistochemistry (IHC) with a novel anti-H. pylori monoclonal antibody that recognizes lipopolysaccharides. Fresh gastric lymph nodes were used to culture for H. pylori. In 46 patients with H. pylori in the stomach, the bacterium was found in the lymph nodes from 21 patients by culture, 37 patients by PCR, and 29 patients by IHC. H. pylori captured by macrophages was found in the lamina propria of 39 patients. In the lymph nodes, the bacterium was found in many macrophages and a few interdigitating dendritic cells at the paracortical areas. H. pylori was also found in the intracellular canaliculi of parietal cells in 21 patients, but intracytoplasmic invasion into gastric epithelial cells was not identified. When compared to the commercially available anti-H. pylori antibodies, the novel antibody showed the highest sensitivity to detect H. pylori-positive macrophages, whereas no difference was found for H. pylori in the mucous layer. The H. pylori-positive macrophages in the lamina propria correlated with chronic gastritis as well as translocation of such cells to the lymph nodes. These results suggest that H. pylori-induced gastric epithelial damage allows the bacteria to invade the lamina propria and translocate to the gastric lymph nodes, which may chronically stimulate the immune system. The bacteria captured by macrophages, whether remaining alive or not, may contribute to the induction and development of H. pylori-induced chronic gastritis.

Global environmental changes are recently occurring faster than any other time. The resulting combinations of natural and anthropogenic disturbances are strongly affecting coral reef communities. Coral damage can be caused by both abiotic factors (temperature, sedimentation, nutrients inputs, ultraviolet radiation) and biotic factors (predation, overgrowth of algae, infectious diseases). These factors acting mostly in synergy had resulted in worldwide coral reef deterioration. Coral bleaching is the most impacting process that is affecting coral survival under elevated sea surface temperature and high irradiance scenario. Bleaching is well known to occur around the world; however, its mechanism is not well understood. This is due to the high complexity of the “coral holobiont” (coral in symbiosis with its zooxanthellae and a microbial community that maintains a delicate balance to keep the coral health). In this chapter a novel point of view of bleaching mechanism using micro/nano-size scales is presented: (1) the study of pigment dynamics during thermal-induced bleaching revealed that bleaching is a detoxification strategy to avoid the formation of reactive oxygen species (ROS), (2) the synergistic action of thermal stress with pathogenic bacteria exacerbates the bleaching process, and (3) the synergistic effect of thermal stress in a nitrate-enriched environment can impede the recovery of corals after a bleaching event, turning the corals to be more susceptible to other environmental or anthropogenic stressors.

Effects of nutrients on the size and composition of phytoplankton and the resulting changes in ecosystem structure were investigated using incubations of unaltered deep-sea waters from 400 m and 700 m depths of Suruga Bay Japan. This new approach allowed us to observe the succession of phytoplankton communities from conditions of high to low nitrogen availability and explore the effects of N source and availability on the composition and size of diatoms under low grazing pressure. Diatoms dominated the phytoplankton biomass throughout the duration of experiments in both incubations. However, among diatoms, the genus Thalassiosira dominated the abundance and biomass when nutrients were replete, and the genus Chaetoceros dominated the abundance and biomass when nitrate became depleted. Our results show that diatoms can dominate other phytoplankton groups not only under nutrient replete conditions, but also (particularly for Chaetoceros ) under low concentrations of recycled nitrogen if silica and phosphate are not deficient. Diatoms 15 μm dominated the biomass in the 400 m incubations during the formation of blooms under nitrate-replete conditions. After nitrate depletion, diatoms > 15 μm dominated the biomass in both incubations. Our results showed that availability of N determined the composition of diatoms if Si and P are not deficient. However, for size the results showed that N source and availability did not determine the size of phytoplankton. Our results also showed that the molar ratio of Si:N uptake was

The distribution of corals in Japan covers a wide range of latitudes, encompassing tropical to temperate zones. However, coral communities in temperate zones contain only a small subset of species. Among the parameters that determine the distribution of corals, temperature plays an important role. We tested the resilience to cold stress of three coral species belonging to the genus Acropora in incubation experiments. Acropora pruinosa, which is the northernmost of the three species, bleached at 13 °C, but recovered once temperatures were increased. The two other species, A. hyacinthus and A. solitaryensis, which has a more southerly range than A. pruinosa, died rapidly after bleaching at 13 °C. The physiological effects of cold bleaching on the corals included decreased rates of photosynthesis, respiration and calcification, similar to the physiological effects observed with bleaching due to high temperature stress. Contrasting hot bleaching, no increases in antioxidant enzyme activities were observed, suggesting that reactive oxygen species play a less important role in bleaching under cold stress. These results confirmed the importance of resilience to cold stress in determining the distribution and northern limits of coral species, as cold events causing coral bleaching and high mortality occur regularly in temperate zones.

Propionibacterium acnes is one of the most commonly implicated etiologic agents of sarcoidosis. We previously reported a complete genome sequence of the C1 strain of P. acnes as a clinical isolate from subcutaneous granulomatous inflammatory lesions in a patient with sarcoidosis. In the present study, we initially searched for genetic profiles specific to the C1 strain by core genome analysis and multiple genome alignment with database sequences from 76 and 9 P. acnes strains, respectively. The analysis revealed that the C1 strain was phylogenetically independent and carried an 18.8-kbp transposon sequence unique to the sarcoid isolate. The unique composite transposon comprised a novel insertion sequence and extrinsic genes from bacteria other than P. acnes. Multilocus sequence typing using 24 sarcoid and 36 non-sarcoid isolates revealed a total of 28 sequence types (STs), including ST26, which was most frequently found without specificity for sarcoid isolates. All 13 ST26 isolates exhibited cell-invasiveness and were confirmed to carry the novel insertion sequence and 4 of the 27 extrinsic CDSs in the transposon, with one exception. ST26 of P. acnes with the composite transposon is the most unique strain detected to date and should be further examined as a causative strain of sarcoidosis.

To test the hypothesis that different phytoplankton cellular components undergo distinct decomposition processes, we examined bacterial decomposition of extracted dissolved organic matter (EX-DOM) and cell debris from the diatom (Skeletonema costatum) using long-term incubation experiments. The decay rates indicated that EX-DOM is extremely bioreactive (1.2-1.7 d-1), and that particulate organic carbon (POC) from cell debris is resistant to rapid microbial degradation (0.047-0.055 d-1). In the experiment with grazers, the percentage of accumulated dissolved organic carbon (DOC) in the EX-DOM and cell debris experiments was 5% and 64%, respectively, of the initial DOC concentrations derived from phytoplankton cells. The initial C/N ratio of DOM in the cell debris experiments was 19.2-19.7, higher than those in the EX-DOM experiments (5.5-6.0). The percentage of accumulated DOC in the cell debris experiment with grazers was 35% at day 13. Thereafter DOC accumulated until it reached 64%. Our data indicate that these two distinct phytoplankton-derived organic materials (EX-DOM and cell debris) decay and accumulate differently. These results suggest that structural components of phytoplankton cells may persist as semi-labile POC, and that DOC produced by solubilization of structural components of cell and phytoplankton-derived DOM with high C/N ratios may accumulate as semi-labile DOC in seawater.

The purpose of this study was to clear the fate of organic carbon and nitrogen in seawater and sediment, especially the difference between organic carbon and nitrogen in dissolved and particulate forms and to determine carbon sequestration by seagrass ecosystem. We had carried out four types experiments: the diel variation for 24 hour, and seasonal changes of DOC (Dissolved Organic Carbon)/DON (Dissolved Organic Nitrogen) and POC (Particulate Organic Carbon)/PON (Particulate Organic Nitrogen) in outside and inside of seagrass communities, and the enclosed chamber experiment for evaluation of release of organic matters from seagrass exuadation and the sediment, decomposition experiments in laboratory and in situ. POM (Particulate Organic Matter) of seagrass leaf litter is resistance to the biological degradation under 25°C. (The rate constant of decomposition is 0.0083 d -1 , namely remineralization time is about 120 days.) While POM in water column shows POC is more rapidly consumed to PON, and POC/PON ratio decresed with time of decomposition. DOM in water column shows DON is more rapidly consumed to DOC and DOC/DON ratio increased with time of decomposition. This suggests that rate of solubilization of DON from PON may dominate a rate of remineralization of nutrients in seagrass communities. We developed a kinetic model of the seagrass decomposition process (SDP) changes in POC/PON ratio with time as well as phytoplankton decomposition model (Fujii et al., 2002). The SDP model was adapted to the results of three kinds of decomposition experiments where the initial C/N ratio and the volume ratio of POM and DOM differed by more than a factor of three. The apparent zeroth-order decrease in POC and the constant PON in the first labile decomposition process are well expressed by the SD model, and consequently, the calculated POC/PON ratio shows good agreement with the experimental result. A realistic model for CO 2 sequestration was also developted using net organic production (net organic production=gross organic production-respiration), sedimentation inside seagrass bed and sedimentation at the open ocean (=export flux). We calculated net sequestration of carbon into deep water after 100 years under two different sinking velocities which are for 6 m/d and 24 m/d. These results showed that 120 t-C for 6 m/d and 150 t-C for 24 m/d during 100 years were accumulated into deepwater, suggesting that 1.2 to 1.5 t-C/year for 31 t-C of gross production of seagrass/km 2 are accumulating into deep water. Seagrass ecosystem is very effective as one of the CO 2 sequestration.

Background Propionibacterium acnes is one of the most commonly implicated etiologic agents of sarcoidosis. We screened antigenic proteins from this indigenous bacterium that increase Th1 responses in sarcoidosis patients. Methods Antigenic bacterial proteins were screened by probing western blots of P. acnes whole cell lysates with blood plasma samples from 52 sarcoidosis patients and 34 healthy volunteers. Soluble protein antigens from the bands most frequently detected on blotting membranes were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Recombinant proteins were prepared from DNA sequences of the proteins identified by MALDI-TOF/MS and analyzed by immunologic assays. Results MALDI-TOF/MS analysis identified propionyl-CoA carboxylase subunit beta, arginine deiminase (ADI), catalase (KAT), and UDP-N-acetylglucosamine pyrophosphorylase (UAP). Successfully prepared recombinant proteins from ADI, KAT, and UAP provoked humoral and cellular immune responses in mice immunized with P. acnes when measured by enzyme-linked immunosorbent assay for serum antibodies and enzyme-linked immunospot assay for interferon (IFN)-γ-secreting cells (ELISPOT IFN-γ assay) with lymph node cells. Plasma IgG and IgA titers to KAT and UAP were significantly higher in sarcoidosis patients than in healthy volunteers. When Th1 immune responses to ADI, KAT, and UAP were measured by ELISPOT IFN-γ assay with peripheral blood mononuclear cells from 12 sarcoidosis patients, 13 other pneumonitis patients, and 11 healthy volunteers, only the KAT protein provoked a significantly higher response in sarcoidosis patients ( p =0.0032). Conclusion These results suggest that P. acnes KAT is an antigen that provokes allergic Th1 immune responses in sarcoidosis patients.

Mycobacterium tuberculosis can survive for a long time in the body of a healthy host, causing no symptoms after primary infection. Years or decades later, M. tuberculosis can be reactivated in some carriers, leading to symptoms of post-primary tuberculosis. During the asymptomatic period, M. tuberculosis is believed to survive in old foci of infection, yet these foci are uniformly negative by culture and acid-fast staining, which are conventional methods to detect mycobacteria. The precise location and amount of M. tuberculosis in these foci, therefore, are not well understood. Here we report that granulomatous lesions with calcification, a well-known characteristic of old tubercles, contained considerable amounts of M. tuberculosis genomes and cell wall components. These calcified lesions exhibited little inflammation but all were positive for M. tuberculosis by real-time polymerase chain reaction and by immunohistochemistry with a novel monoclonal antibody against lipoarabinomannan, a cell wall component of mycobacteria. We found many lipoarabinomannan-containing granules in the necrotic areas of the lesions that were negative by Ziehl-Neelsen staining but included some bacillus-like structures. The risk of reactivation of latent tuberculosis from these calcified granulomatous lung lesions should be considered.

The effects of elevated temperature and high pCO2 on the metabolism of Galaxea fascicularis were studied with oxygen and pH microsensors. Photosynthesis and respiration rates were evaluated from the oxygen fluxes from and to the coral polyps. High-temperature alone lowered both photosynthetic and respiration rates. High pCO2 alone did not significantly affect either photosynthesis or respiration rates. Under a combination of high-temperature and high-CO2, the photosynthetic rate increased to values close to those of the controls. The same pH in the diffusion boundary layer was observed under light in both (400 and 750 ppm) CO2 treatments, but decreased significantly in the dark as a result of increased CO2. The ATP contents decreased with increasing temperature. The effects of temperature on the metabolism of corals were stronger than the effects of increased CO2. The effects of acidification were minimal without combined temperature stress. However, acidification combined with higher temperature may affect coral metabolism due to the amplification of diel variations in the microenvironment surrounding the coral and the decrease in ATP contents.

Sarcoidosis likely results from the exposure of a genetically susceptible subject to an environmental agent, possibly an infectious one. Mycobacterial and propionibacterial organisms are the most commonly implicated potential etiologic agents. Propionibacterium acnes is the only microorganism, however, found in sarcoid lesions by bacterial culture. To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger-factor protein (TIG antibody). We examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients. Immunohistochemistry with PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. In nongranulomatous areas, small round bodies detected by PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. Large-spheroidal acid-fast bodies, Hamazaki-Wesenberg bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these Hamazaki-Wesenberg bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body. The high frequency and specificity of P. acnes, detected by PAB antibody within sarcoid granulomas, indicates that this indigenous bacterium might be the cause of granuloma formation in many sarcoid patients. © 2012 USCAP, Inc. All rights reserved.

Background Propionibacterium acnes and Mycobacterium tuberculosis have emerged as probable candidates responsible for sarcoidosis. This study was conducted to investigate the Th1/Th17 responses elicited by these pathogens in sarcoidosis and to clarify the causative role of these pathogens. Methods Peripheral blood mononuclear cells (PBMCs) obtained from patients with sarcoidosis and from healthy volunteers were, respectively, co-cultured with viable P. acnes , with Bacille de Calmette et Guerin (BCG) as a viable M. tuberculosis complex, and with the early secretory antigenic target (ESAT)-6. Th1 cytokine production was measured using RT-PCR and enzyme-linked immunospot (ELISPOT) assays, and interleukin (IL)-17 mRNA expression was measured by RT-PCR. Results IL-2 secretion from PBMCs after stimulation with P. acnes was significantly higher in patients with sarcoidosis than in the controls. Similarly, IL-2 and IL-12 mRNA expression after stimulation with P. acnes was significantly higher in PBMCs from patients with sarcoidosis than in PBMCs from controls. In contrast, IL-17 mRNA expression was significantly lower in PBMCs from patients with sarcoidosis than in PBMCs from controls. No significant differences between the groups were observed in the responses to stimulation with BCG or ESAT-6. Conclusion Sarcoidosis may arise from an imbalance of Th1/Th17 immune responses against viable P. acnes , but not M. tuberculosis complex.

Numerous publications have reported the presence of periodontopathogenic bacteria in peripheral and central vascular lesions. However, it is unclear how this bacterial translocation occurs. The objective of this study was to investigate whether periodontopathic bacteria are translocated to lymph nodes proximal to the oral cavity. Obtaining lymph node samples is not ethically feasible unless they are excised as part of the surgical management of patients with cancer. This study analyzed formalin-fixed and paraffin-embedded lymph nodes, histologically negative for cancer cell invasion, that were excised from 66 patients with histories of head and neck cancer. Real-time PCR was performed to amplify the 16S ribosomal DNA fragments from Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Prevotella intermedia. The relationship between bacterial detection and cancer severity, gender, and the use of anti-cancer therapy was examined by Fisher's exact test. P. gingivalis, T. forsythia, and P. intermedia were present in 17%, 8%, and 8% of the samples of submandibular and submental lymph nodes, respectively. There were no significant relationships between bacterial detection and the cancer disease status, patient gender or use of anticancer therapy. According to these data, it appears that the translocation of periodontopathic bacteria may occur via lymphatic drainage, irrespective of the cancer disease status, gender or anticancer therapy.

Cyanobacteria have dominated marine environments and have been reef builders on Earth for more than three million years (myr). Cyanobacteria still play an essential role in modern coral reef ecosystems by forming a major component of epiphytic, epilithic, and endolithic communities as well as of microbial mats. Cyanobacteria are grazed by reef organisms and also provide nitrogen to the coral reef ecosystems through nitrogen fixation. Recently, new unicellular cyanobacteria that express nitrogenase were found in the open ocean and in coral reef lagoons. Furthermore, cyanobacteria are important in calcification and decalcification. All limestone surfaces have a layer of boring algae in which cyanobacteria often play a dominant role. Cyanobacterial symbioses are abundant in coral reefs; the most common hosts are sponges and ascidians. Cyanobacteria use tactics beyond space occupation to inhibit coral recruitment. Cyanobacteria can also form pathogenic microbial consortia in association with other microbes on living coral tissues, causing coral tissue lysis and death, and considerable declines in coral reefs. In deep lagoons, coccoid cyanobacteria are abundant and are grazed by ciliates, heteroflagellates, and the benthic coral reef community. Cyanobacteria produce metabolites that act as attractants for some species and deterrents for some grazers of the reef communities.

Behavior of dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) in coral reef waters in relation with biological processes was studied with incubation experiments and field observations in May 2008 and 2009 at the fringing reef of Sesoko Island, Okinawa, Japan. Reef sea water (RSW) and coral mucus added RSW collected from Acropora digitifera (AcrRSW) and Montipora digitata (MonRSW) were incubated for one day in situ and then for 77 days in the laboratory under dark condition . The results indicated that the behavior of DON was different compared to that of DOC in RSW and mucus added (AcrRSW and MonRSW) during dark incubation. Concentration of DON increased from 8.3 µM to 11.8 µM for AcrRSW and 4.0 µM to 15.4 µM for MonRSW during dark incubation period. The increasing rates for DON in AcrSRW and MonRSW were 0.05 µM day -1 and 0.1 µM day -1 respectively. On the other hand DOC concentration decreased from 129.0 µM to 75.0 µM for AcrRSW and 75.1 µM to 64.7 µM for MonRSW, with decreasing rates of 0.7 µM day -1 and 0.1 µM day -1 respectively. We assume that the increase of DON may be determined by difference between rates of inputs of organic matter mainly from mucus and rate of degradation of dissolved organic matter in the water column. These results suggest that recycling of DON is slow than that of DOC in coral reef ecosystem.

Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert-butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K(m) value was estimated to be 44.4 microM for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS-PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30-90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions.

The purpose of this study is to find a method of cooking natto that prevents the appearance of high-plasma vitamin K concentrations after the consumption of natto, so that patients taking warfarin can benefit from eating natto. Five cooking methods were examined to determine which could most effectively decrease the count of the living Bacillus subtilis in natto. Volunteers ate natto or treated natto, and their plasma vitamin K level was measured at 5, 8, 24 and 48 h thereafter. One gram of natto contained 9.7+/-0.1 Log cfu/mL of Bacillus subtilis. Boiling significantly reduced the Bacillus subtilis count to 5.1+/-0.3 Log cfu/mL, and concomitantly reduced the content of menaquinone-7 (MK-7), which is a form of vitamin K synthesized by Bacillus subtilis, from 660.40+/-65.32 ng/mL to 78.50+/- 11.12 ng/mL. Untreated natto increased the MK-7 concentration in blood from 1.86+/-1.51 ng/mL to 14.54+/-4.12 ng/mL at 5 h after intake, and the MK-7 concentration remained elevated at 8, 24 and 48 h (7.29+/-2.20, 6.97+/-2.60, and 5.37+/-1.94 ng/mL, respectively). In contrast, boiled natto increased plasma MK-7 only mildly (from 1.61+/-1.11 to 4.02+/-0.82 ng/ mL at 5 h) and the concentration remained relatively stable up to 48 h (3.46+/-0.83, 4.22+/-1.51 and 2.77+/-0.75 ng/mL at 8, 24 and 48 h, respectively). In conclusion, boiled natto did not cause a marked increase in the plasma concentration of vitamin K in subjects who consumed it. Thus, patients on warfarin may be able to eat boiled natto without ill effects.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. NOD2 mutations have been shown to predispose to granulomatous diseases, including Crohn's disease, Blau syndrome, and early-onset sarcoidosis, but not to adult sarcoidosis. We found that intracellular Propionibacterium acnes, a possible causative agent of sarcoidosis, activated NF-κB in both NOD1- and NOD2-dependent manners. Systematic search for NOD1 gene polymorphisms in Japanese sarcoidosis patients identified two alleles, 796G-haplotype (156C, 483C, 796G, 1722G) and 796A-haplotype (156G, 483T, 796A, 1722A). Allelic discrimination of 73 sarcoidosis patients and 215 healthy individuals showed that the frequency of 796A-type allele was significantly higher in sarcoidosis patients and the ORs were significantly elevated in NOD1-796G/A and 796A/A genotypes (OR [95% CI] = 2.250 [1.084, 4.670] and 3.243 [1.402, 7.502], respectively) as compared to G/G genotype, showing an increasing trend across the 3 genotypes (P = 0.006 for trend). A similar association was found when 52 interstitial pneumonia patients were used as disease controls. Functional studies showed that the NOD1 796A-allele was associated with reduced expression leading to diminished NF-κB activation in response to intracellular P. acnes. The results indicate that impaired recognition of intracellular P. acnes through NOD1 affects the susceptibility to sarcoidosis in the Japanese population.

Publisher Summary This chapter evaluates the effectiveness of an artificial marine structure as an upwell-generator to enhance oceanic CO 2 sequestration in the coastal areas of Japan. Results suggest that the processes responsible for vertical mixing between shallow waters and bottom waters were vertical shear of currents, internal wave breaking, and entrainment. It also found evidence that neap tides may enhance the mixing. The upwelling generated by the artificial marine structure, increased nutrient supply to the shallow layer. Coccoid pico-cyanobacteria represented about 25% to 30% of the total biomass produced during incubation. Diatoms of small size were abundant, among which Chaetoceros compressum and other Chaetoceros species predominated. Net organic production estimated over the year is 470 gC m -2 yr -1 using 1300 mgC m -2 day -1 . This value is the same order of magnitude as net organic production in temperate forests and mangroves. This, together with the contribution from forestation, could help enable Japan to reach its CO 2 -reduction target as set in the Kyoto Protocol. It is concluded that the use of marine biota, natural energy and this artificial upwelling system contributes effectively to increased CO 2 fixation.