pO2-dependent NO production determines OPPC activity in macrophages

Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O₂, L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO₂ on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon γ (IFNγ) at pO₂ ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO₂ > 0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O₂, without decreasing NOx production. These results are the first to investigate the effect of pO₂ on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia.     

Historic fuel wood use in the Galápagos Islands: identification of charred remains

Charcoal fragments from five historic campsite locations in the Galápagos Islands were identified and radiocarbon dated to investigate postulated early human presence in the archipelago, historic fuel wood collection patterns and the resultant impact on native vegetation. A variety of taxa and fuel types were revealed to be present in the charcoal assemblages, indicating geographically driven rather than species-specific methods of collection. Historic anthropogenic impact was therefore spread amongst woody taxa in the lowland plant communities, with severity dependent on proximity to campsite location. All charred remains were found to date from within the historic period, supporting the preponderance of archaeological evidence indicating that human presence did not begin in Galápagos until after European discovery.     

Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish

After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.     

Archaea, Bacteria, and Algal Plastids Associated with the Reef-Building Corals Siderastrea stellata and Mussismilia hispida from Búzios, South Atlantic Ocean, Brazil

Reef-building corals may be seen as holobiont organisms, presenting diverse associated microbial communities. Best known is the symbiotic relationship with zooxanthellae, but Archaea, Bacteria, fungi, viruses, and algal plastids are also abundant. Until now, there is little information concerning microbial communities associated with Brazilian corals. The present study aims to describe the diversity of Archaea, Bacteria, and eukaryotic algal plastid communities associated with two sympatric species, Siderastrea stellata and Mussismilia hispida, from Southeastern Brazil, using 16S rRNA gene libraries. Since corals present a high number of other associated invertebrates, coral barcoding (COI) was performed to confirm the exclusive occurrence of coral DNA in our samples. Our analysis yielded 354 distinct microbial OTUs, represented mainly by novel phylotypes. Richness (Chao1 and ACE) and diversity (H') estimations of the microbial communities associated with both species were high and comparable to other studies. Rarefaction analyses showed that microbial diversity of S. stellata is higher than that of M. hispida. Libshuff comparative analyses showed that the highest microbial community similarity between the two coral species occurred in the bacterial libraries, while archaeal and plastidial communities were significantly different. Crenarchaeota dominated archaeal communities, while Proteobacteria was the most abundant bacterial phylum, dominated by alpha-Proteobacteria. Plastids were also represented by novel phylotypes and did not match with any 16S rRNA sequences of Cyanobacteria and zooxanthellae from GenBank. Our data improves the pool of available information on Brazilian coral microbes and shows corals as sources of diverse prokaryotic and picoeukaryotic communities.     

Mouse model of skeletal muscle adiposity: A glycerol treatment approach

Fat cell accumulation in skeletal muscle is a major characteristic of various disorders, such as obesity, sarcopenia and dystrophies. Moreover, these fat cells could be involved in muscle homeostasis regulation as previously described for adipocytes in bone marrow. Despite recent advances on the topic, no clearly characterized mouse model is currently available to study fat accumulation within skeletal muscle. Here, we report a detailed characterization of a mouse model of skeletal muscle fat cell accumulation after degeneration induced by intra-muscular injection of glycerol. Information is provided on the kinetics of degeneration/fat deposition, including the quantity of fat deposited based on various parameters such as glycerol concentration, age, sex and strain of mice. Finally, these fat cells are characterized as true white adipocytes morphologically and molecularly. Our study shows that the mouse adipocyte accumulation within skeletal muscle after glycerol degeneration is a reproducible, transposable and easy model to use. This mouse model should allow a more comprehensive understanding of the impact of adipocyte accumulation in skeletal muscle pathophysiology.     

Tumor Immunity against a Simian Virus 40 Oncoprotein Requires CD8+ T Lymphocytes in the Effector Immune Phase

The required activities of CD4⁺ T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8⁺ T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8⁺ T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-γ), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8⁺ T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8⁺ T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8⁺ T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.Determining the immunologic mechanisms involved in antitumor responses can provide valuable insight into developing and formulating appropriate immunotherapeutic strategies against a range of human cancers (25). Cell-mediated immunity involving CD8⁺ T lymphocytes is generally regarded as the primary response to utilize due to its potent and efficient cytotoxicity against tumor cell targets in vitro and in animal models (10). Indeed, the proof of concept of this approach is best characterized by specialized conditioning protocols that involve autologous transfer of tumor infiltrating lymphocytes (TILs) in metastatic melanoma patients, with objective responses that approximate 70% (8). However, the efficacy of TILs harvested from additional cancer types have been less than effective, and additional strategies, such as genetic modification of peripheral blood mononuclear cells, are being explored to improve and extend the approach of cytotoxic T-lymphocyte (CTL) immunotherapy clinically (33, 46).The roles of immune components such as CD4⁺ T cells and antibody have been given less attention within the context of promoting tumor immunity against a range of tumor antigens. For example, the ability of CD4⁺ T cells to activate humoral immunity can lead to antitumor responses that involve antibody-dependent cell-mediated cytotoxicity (ADCC) (17). In this scenario, antibody binds its targeted antigen and effectors such as natural killer (NK) cells lyse tumorigenic cells through interaction with the Fc region of the bound antibody. The efficacy of ADCC has been realized in scenarios involving breast cancer and non-Hodgkin''s lymphoma, for example, and to date, the only FDA-approved immunologic treatments against these malignancies involve antibody-based therapies (5).The concurrent roles of antibody—with specific emphasis on ADCC—and CD8⁺ T-cell immunity within the context of tumor immunity have not been widely reported. Several recent studies have commented on the ability of antibody-bound tumor cells, particularly as a whole tumor cell-dendritic cell (DC) vaccination approach, to initiate CTL activity by engaging DCs through Fc receptors (9, 19, 34). However, to our knowledge, the mechanistic aspects of ADCC (e.g., NK-mediated lysis) promoting CD8⁺ T-cell activity have been explored in relatively few studies (27, 41). From an immunotherapeutic standpoint, it may be preferable in certain settings to induce both the humoral and cell-mediated arms of the immune system to offset the progression of tumor cell growth and dissemination. Namely, these strategies could include active or passive approaches to first effectively induce ADCC in response to a tumor antigen, which would promote CTL activity against additional tumor targets through cross-presentation.Our laboratory has been involved in determining the immunologic mechanisms of tumor immunity induced by the virally encoded tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag). The mechanistic aspects of SV40 Tag-induced tumor immunity have been examined within an experimental murine model of pulmonary metastasis. To date, CD4⁺ T cells and SV40 Tag-specific antibody have been implicated as required immune components within this murine system in order to achieve complete systemic tumor immunity (18). These studies demonstrated that during the course of immunization with SV40 Tag (i.e., the induction-phase response), CD4⁺ T cells were required to induce an SV40 Tag humoral response. The specific role of the antibody response against an experimental tumor cell challenge was observed to involve ADCC-mediated clearance pathways (4, 23).In the present study, we further characterize the immunologic response to SV40 Tag immunization by observing the necessary immune cell components following experimental challenge (i.e., the effector-phase response) with a tumor cell line expressing SV40 Tag. With the development of an SV40 Tag antibody response following SV40 Tag immunization in vivo, CD8⁺ T-cell depletion during the effector phase resulted in the formation of lung tumor foci and decreased survival not observed with the abrogation of CD4⁺ T cells. SV40 Tag-immunized mice also displayed a heightened Th1 response and CD8⁺ CTL activity after experimental tumor cell challenge, as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. In all, these data indicate that CD8⁺ T cells mediate tumor immunity following antibody activation in response to the tumor-specific antigen SV40 Tag. We hypothesize that CD8⁺ T-cell activity is initiated due to cross-presentation mechanisms as a result of ADCC activity against SV40 Tag. We are not aware of another published report that formulates a role for ADCC activity against a viral oncoprotein in this manner in order to engage CD8⁺ T-cell activation.SV40 Tag has been reported to be expressed in a number of human cancers, including malignant pleural mesothelioma and non-Hodgkin''s lymphoma, although a causal link between SV40 infection and tumorigenesis is uncertain (11, 24, 35). The results of this study have direct implications for the construction of an appropriate immunotherapeutic strategy for patients suffering malignancies expressing the SV40 Tag tumor-specific antigen.     

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome encodes several smaller open reading frames (ORFs) located in the 3′ region of the genome that are predicted to express eight novel proteins termed accessory proteins. The accessory proteins are designated ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b and range in size from 39 to 274 amino acids (35, 50). These SARS-CoV-specific ORFs are not present in other coronaviruses and do not display significant homology with any known proteins in the NCBI database. Five of these are predicted to code for polypeptides of greater than 50 amino acids (35, 50). Antibodies reactive against all of the SARS-CoV proteins have been detected in sera isolated from SARS patients, indicating that these proteins are expressed by the virus in vivo (7, 9, 17-19, 45, 59). Expression of three of the ORF proteins has been demonstrated during infection using protein-specific antibodies and include the ORFs 3a, 6, and 7a (12, 37, 41, 60). Six of the eight group-specific ORFs, including ORFs 3a, 3b, 6, 7a, 7b, and 9b, were deleted from recombinant SARS-CoV and shown to be dispensable for in vitro and in vivo replication (66).Related coronaviruses also encode unique accessory proteins in the 3′ region of the genome, often referred to as group-specific ORFs. Similar to SARS-CoV, several of these proteins are dispensable for viral replication. Murine hepatitis virus (MHV) expresses accessory proteins ORFs 2a, 4, and 5a. A recombinant virus in which ORF 2a was deleted replicated normally in vitro but caused attenuated disease in vivo (55). Deletion of the group-specific ORF 7 in porcine coronavirus TGEV also results in reduced replication and virulence in vivo despite normal replication in vitro (38). Similarly, in feline infectious peritonitis virus (FIPV), group-specific proteins are dispensable for replication in cell culture but contribute to pathogenesis in vivo (20). Thus, while the SARS-CoV group specific proteins are unnecessary for in vitro and in vivo replication, their expression may underlie the devastating pathology associated with SARS disease. Detailed characterization of these novel proteins may contribute to a better understanding of SARS pathogenesis and host-virus interactions.The ORF 3a protein is expressed from subgenomic RNA3, which contains the 3a and 3b ORFs (35, 50). The 3a protein, which is the largest group-specific SARS-CoV accessory protein at 274 amino acids, has been reported to localize to the Golgi apparatus, the plasma membrane, and intracellular vesicles of unknown origin (67, 68). The protein is efficiently transported to the cell surface and is also internalized during the process of endocytosis (60).The mechanism of SARS-CoV-induced cell death has been investigated by several groups. Studies to date have used overexpression of individual SARS-CoV ORFs to evaluate their intrinsic cytotoxicity. Using this approach, the following proteins have been reported to cause apoptosis: the 3CL-like protease; spike; ORFs 3a, 3b, and 7a; and the envelope (E), membrane (M), and nucleocapsid (N) proteins (23, 31, 32, 36, 46, 58, 61, 65, 69). However, since all of these reports utilize overexpression of individual proteins, it is unclear whether these effects may be attributable to high, nonphysiological levels of protein and whether they occur during infection. Analysis of recombinant viruses with specific mutations or deletions is necessary to determine the relative contribution of these proteins to the cytotoxicity of SARS-CoV during infection (63). Therefore, the cytotoxic component(s) of SARS-CoV have not been fully defined.Here, we have investigated the function of the ORF 3a protein in the context of SARS-CoV infection and by overexpression. We confirm that ORF 3a contributes to SARS-CoV cytotoxicity using a recombinant strain deficient for expression of ORF 3a. While characterizing this deficient strain, we observed that SARS-CoV-induced vesicle formation, a feature that has been documented in cells from infected SARS patients, is dependent on ORF 3a. Furthermore, we observed that SARS-CoV infection causes Golgi fragmentation by ORF 3a. Additional characterization of 3a in transfected cells revealed that the protein colocalizes with markers of the trans-Golgi network (TGN) and late endosomal pathways and causes an accumulation of these vesicles. Finally, we report that Arf1 overexpression rescued SARS-CoV or 3a-induced Golgi fragmentation, suggesting that the ORF 3a protein may perturb Arf1-mediated vesicle trafficking.