Intracellular Growth Inhibition and Host Immune Modulation of 3-Amino-1,2,4-triazole in Murine Brucellosis.

Catalase, an antioxidant enzyme widely produced in mammalian cells and bacteria, is crucial to mitigating oxidative stress in hostile environments. This function enhances the intracellular survivability of various intracellular growth pathogens, including Brucella (B.) abortus. In this study, to determine whether the suppression of catalase can inhibit the intracellular growth of B. abortus, we employed 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, in both RAW 264.7 macrophage cells and an ICR mouse model during Brucella infection. The intracellular growth assay indicated that 3-AT exerts growth-inhibitory effects on B. abortus within macrophages. Moreover, it contributes to the accumulation of reactive oxygen species and the formation of nitric oxide. Notably, 3-AT diminishes the activation of the nucleus transcription factor (NF-κB) and modulates the cytokine secretion within infected cells. In our mouse model, the administration of 3-AT reduced the B. abortus proliferation within the spleens and livers of infected mice. This reduction was accompanied by a diminished immune response to infection, as indicated by the lowered levels of TNF-α, IL-6, and IL-10 and altered CD4+/CD8+ T-cell ratio. These results suggest the protective and immunomodulatory effects of 3-AT treatment against Brucella infection.

Comparative Genome and Evolution Analyses of an Endangered Stony Coral Species Dendrophyllia cribrosa Near Dokdo Islands in the East Sea.

Stony corals often harbor intracellular photosynthetic dinoflagellate algae that receive dissolved inorganic nutrients. However, Dendrophyllia cribrosa is a nonsymbiotic stony coral distributed in the western Pacific. We assembled a chromosome-level D. cribrosa genome using PacBio and Hi-C technologies. The final assembly was 625 Mb, distributed on 14 chromosomes, and contained 30,493 protein-coding genes. The Benchmarking Universal Single-Copy Orthologs analysis revealed a percentage of 96.8 of the metazoan genome. A comparative phylogenetic analysis revealed that D. cribrosa, which lacks symbionts, evolved to acquire cellular energy by expanding genes related to acyl-CoA metabolism and carbohydrate transporters. This species also has expanded immune-related genes involved in the receptor protein tyrosine kinase signaling pathway. In addition, we observed a specific expansion of calcification genes, such as coral acid-rich proteins and carbonic anhydrase, in D. cribrosa. This high-quality reference genome and comparative analysis provides insights into the ecology and evolution of nonsymbiotic stony corals.

Respiration of the sea urchin Mesocentrotus nudus in response to large temperature fluctuations.

Some subtidal habitats may experience extremely large diel temperature fluctuations. To explore the potential of subtidal animals to regulate their metabolic processes, we investigated how the oxygen consumption rate (MO2) of the sea urchin Mesocentrotus nudus changes in response to extreme temperature fluctuations by mimicking temperature variations recorded at Dokdo Island, Republic of Korea. We compared the MO2 of urchins before and after a temperature fluctuation. MO2 was positively correlated with temperature. There was no change in the mean MO2 values even after exposure to fluctuating temperature. There was no significant difference in mean MO2 between large and small temperature fluctuations. These results indicate that the metabolic activity of M. nudus might be well-adapted to extreme temperature fluctuations. However, given that the temperature coefficient (Q10) values decreased with increasing temperature and Q10 values during the temperature decrease was higher than those during temperature increase, temperature rise may still act as a stressor for these animals.

The complete mitochondrial genome of a Dokdo shrimp, Lebbeus groenlandicus.

Lebbeus groenlandicus is a shrimp species indigenous to the Dokdo islands in the East Sea of Korea. We report the 17,399 bp mitochondrial genome (mitogenome) of the species that consists of 13 protein-coding genes, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region (CR). A maximum-likelihood tree, constructed with 18 prawn and 45 shrimp mitogenomes, confirmed that L. groenlandicus occupies the most basal position within the Caridea infra-order and is closely related to Pandalidae shrimps.

Heat-stress-modulated induction of NF-κB leads to brucellacidal pro-inflammatory defense against Brucella abortus infection in murine macrophages and in a mouse model.

BACKGROUND: Brucella causes a chronic and debilitating infection that leads to great economic losses and a public health burden. In this study, we demonstrated the brucellacidal effect of heat shock mediated by the induction of pro-inflammatory cytokines, reactive oxygen species (ROS) accumulation and apoptosis in murine macrophages and in mice. RESULTS: RAW264.7 cells were incubated at 43 °C, and BALB/c mice were subjected to whole body hyperthermia. The data showed a reduction in bacterial survival in the mice after daily heat exposure. This was accompanied by increased levels of cytokines TNF, IL-6, IL-1ß and IFN-γ in the sera of the mice. Gene expression of NF-κB and inducible nitric oxide production were also induced in the mouse splenic cells. In parallel with the bacterial reduction in the mouse model, an increased bactericidal effect was observed in RAW264.7 cells after exposure to heat stress. In addition, the heat stress increased both the nuclear translocation of NF-κB and the expression of the heat shock proteins HSP70 and HSP90 in murine macrophages. Furthermore, heat exposure induced the increase of pro-inflammatory cytokines, ROS accumulation and apoptosis but did not affect the production of nitric oxide (NO) in macrophages. CONCLUSION: This study demonstrated the induction of innate immune responses by heat stress that significantly reduced the intracellular survival of B. abortus in vitro and in vivo. Transcriptional factor NF-κB, which is a master regulator, could be termed a key activator of heat-induced immunity against Brucella. The increase in the expression and activation of NF-κB in splenic cells and macrophages was followed by enhanced antimicrobial effectors, including cytokines, ROS and NO that may contribute to the reduction of bacterial survival.

Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells.

Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

Bathing Effects of Various Seawaters on Allergic (Atopic) Dermatitis-Like Skin Lesions Induced by 2,4-Dinitrochlorobenzene in Hairless Mice.

We evaluated the preventive effects of four types of seawater collected in Republic of Korea on hairless mice with 2,4-dinitrochlorobenzene- (DNCB-) induced allergic/atopic dermatitis (AD). The anti-inflammatory effects were evaluated by measuring tumor necrosis factor- (TNF-) α and interleukins (ILs). Glutathione (GSH), malondialdehyde (MDA), superoxide anion, and inducible nitric oxide synthase (iNOS) were measured to evaluate the antioxidant effects. Caspase-3 and poly (ADP-ribose) polymerase (PARP) were observed to measure the antiapoptotic effects; matrix metalloproteinase- (MMP-) 9 levels were also evaluated. Mice with AD had markedly higher clinical skin severity scores and scratching behaviors; higher TNF-α and ILs (1ß, 10, 4, 5, and 13) levels; higher MDA, superoxide anion, caspase-3, PARP, and MMP-9 levels; and greater iNOS activity. However, the severity of AD was significantly decreased by bathing in seawaters, but it did not influence the dermal collagen depositions and skin tissue antioxidant defense systems. These results suggest that bathing in all four seawaters has protective effects against DNCB-induced AD through their favorable systemic and local immunomodulatory effects, active cytoprotective antiapoptotic effects, inhibitory effects of MMP activity and anti-inflammatory and antioxidative effects.

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus.

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.

Redundant effects of ketamine on the pathogenesis and severity of Brucella abortus infection.

Brucella abortus is an intracellular bacterium and leading to a serious debilitating disease known as brucellosis. Ketamine is an anesthetic and a sedative that affects the immunomodulatory activities of various immune cells. The current study was to elucidate the role of ketamine in B. abortus infection, focusing on the phagocytic activity and immune response of macrophages. Following incubation of murine macrophages with ketamine, the phagocytosis of B. abortus was markedly reduced compared with the unincubated control. Interestingly, ketamine-incubated cells displayed a decreased intensity of F-actin fluorescence compared with the B. abortus-induced amplification of intensity. Conversely, the intracellular replication of B. abortus within macrophages was notably enhanced by ketamine. Furthermore, the in vivo assessment using a mouse model revealed that continual injections with ketamine led to augmented bacterial burdens in the spleen, which was accompanied by decreased levels of mRNA expression of cytokines in the spleen. The elevations of serum cytokines such as IFN-γ, IL-12 and IL-6, as well as the chemokine MCP-1, were also reduced by ketamine. These findings verify that ketamine suppresses the phagocytic activity and immune response during B. abortus infection. Therefore, the current study might provide novel insights into the potential influences of ketamine on infectious diseases caused by B. abortus, considering the host-pathogen interaction.

Megadraconema cornutum, a new genus and species from Korea, with a discussion of its classification and relationships within the family Draconematidae (Nematoda, Desmodorida) based on morphological and molecular characters.

A new genus and species of Draconematidae Filipjev, 1918, Megadraconema cornutum gen. nov., sp. nov., inhabiting subtidal sediments in Jejudo, Korea is described. Megadraconema cornutum gen. nov., sp. nov. is mainly characterized by a long body (1630-2220 µm), presence of a transverse circle of well-developed papillae-like cuticular protrusions at the base of the lip region, a head capsule with reticular structure of subcuticle, an amphid with a pore-like opening, and an internal, bar-shaped fovea. The diagnosis of the family Draconematidae is emended and a key to genus is provided based on their major differential diagnostic characteristics, summarized in a table. Phylogenetic relationships of all the genera within the Draconematidae are discussed for the first time, based on molecular analyses and morphological features. The phylogenetic position of the new genus and relationships within the family Draconematidae based on analysis of molecular sequence data are examined. Analysis of 18S rRNA gene sequences does not support the currently accepted classification, and indicates paraphyly of the subfamily Draconematinae.

Biological and antibacterial activities of the natural herb Houttuynia cordata water extract against the intracellular bacterial pathogen salmonella within the RAW 264.7 macrophage.

Salmonellosis is a major bacterial zoonosis that causes a variety of disease syndromes, from self-limiting enteritis to fatal infection in animals and food-borne infection and typhoid fever in humans. Recently, the emergence of multidrug-resistant strains of Salmonella sp. has caused more serious problems in public health. The present study investigated the antibacterial effects of Houttuynia cordata water extract (HCWE) against murine salmonellosis. In RAW 264.7 cells, there was no detectable cytotoxic effect of HCWE at any concentration between 25 and 100 microg/ml after 8-h incubation. The antibacterial activity of HCWE was then examined in a Salmonella enterica serovar (Salmonella typhimurium), and was found to increase in a dose-dependent manner at concentrations from 25 to 100 microg/ml during 8-h incubation. HCWE also affected RAW 264.7 cells including morphologic change and bacterial uptake, but there was no significant difference in bacterial replication in RAW 264.7 cells. With HCWE alone, nitric oxide (NO) production by RAW 264.7 cells did not increase, but when RAW 264.7 cells were infected by S. typhimurium, with or without HCWE, NO production with HCWE was 2-fold higher than that without HCWE. Treatment with HCWE did not affect inducible NO synthase (iNOS) mRNA expression by RAW 264.7 cells, but when RAW 264.7 cells with HCWE were infected by S. typhimurium, iNOS mRNA expression was increased during 8-h incubation. Furthermore, HCWE showed virulence reduction effects in S. typhimurium-infected BALB/c mice. After a lethal dose of S. typhimurium, the mortality rate in the HCWE untreated group was 100% at 7 d, but the HCWE 25, 50, and 100 microg/ml groups survived until 11, 17, and 23 d, respectively. These data suggest that HCWE is stable and beneficial in the treatment of bacterial infection including intracellularly replicating pathogens and may solve antimicrobial misuse and overuse.