Treatment with Polyethylene Glycol-Conjugated Fungal D-Amino Acid Oxidase Reduces Lung Inflammation in a Mouse Model of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency wherein phagocytes are unable to produce reactive oxygen species (ROS) owing to a defect in the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) complex. Patients with CGD experience bacterial and fungal infections and excessive inflammatory disorders. Bone marrow transplantation and gene therapy are theoretically curative; however, residual pathogenic components cause inflammation and/or organic damage in patients. Moreover, antibiotic treatments may not help in preventing excessive inflammation due to the residual presence of fungal cell wall ß-glucan. Thus, better treatment strategies against CGD are urgently required. Polyethylene glycol-conjugated recombinant porcine D-amino acid oxidase (PEG-pDAO) supplies ROS to defective NADPH oxidase in neutrophils of patients with CGD, following which the neutrophils regain bactericidal activity in vitro. In this study, we employed an in vivo nonviable Candida albicans (nCA)-induced lung inflammation model of gp91-phox knockout CGD mice and supplied novel PEG conjugates of Fusarium spp. D-amino acid oxidase (PEG-fDAO), as it exhibits higher enzyme activity than PEG-pDAO. The body weight, lung weight, and lung pathology were evaluated using three experimental strategies with the in vivo lung inflammation model to test the efficacy of the ROS-generating enzyme replacement therapy with PEG-fDAO. The lung weight and pathological findings suggest the condition was ameliorated by administration PEG-fDAO, followed by intraperitoneal injection of D-phenylalanine or D-proline. Although a more precise protocol is essential, these data reveal the targeted delivery of PEG-fDAO to the nCA-induced inflammation site and show that PEG-fDAO can be used to treat inflammation in CGD in vivo.

Comparative Study of the Gut Microbiota Among Four Different Marine Mammals in an Aquarium.

Despite an increasing appreciation in the importance of host-microbe interactions in ecological and evolutionary processes, information on the gut microbial communities of some marine mammals is still lacking. Moreover, whether diet, environment, or host phylogeny has the greatest impact on microbial community structure is still unknown. To fill part of this knowledge gap, we exploited a natural experiment provided by an aquarium with belugas (Delphinapterus leucas) affiliated with family Monodontidae, Pacific white-sided dolphins (Lagenorhynchus obliquidens) and common bottlenose dolphin (Tursiops truncatus) affiliated with family Delphinidae, and Cape fur seals (Arctocephalus pusillus pusillus) affiliated with family Otariidae. Results show significant differences in microbial community composition of whales, dolphins, and fur seals and indicate that host phylogeny (family level) plays the most important role in shaping the microbial communities, rather than food and environment. In general, the gut microbial communities of dolphins had significantly lower diversity compared to that of whales and fur seals. Overall, the gut microbial communities were mainly composed of Firmicutes and Gammaproteobacteria, together with some from Bacteroidetes, Fusobacteria, and Epsilonbacteraeota. However, specific bacterial lineages were differentially distributed among the marine mammal groups. For instance, Lachnospiraceae, Ruminococcaceae, and Peptostreptococcaceae were the dominant bacterial lineages in the gut of belugas, while for Cape fur seals, Moraxellaceae and Bacteroidaceae were the main bacterial lineages. Moreover, gut microbial communities in both Pacific white-sided dolphins and common bottlenose dolphins were dominated by a number of pathogenic bacteria, including Clostridium perfringens, Vibrio fluvialis, and Morganella morganii, reflecting the poor health condition of these animals. Although there is a growing recognition of the role microorganisms play in the gut of marine mammals, current knowledge about these microbial communities is still severely lacking. Large-scale research studies should be undertaken to reveal the roles played by the gut microbiota of different marine mammal species.

Aging- and obesity-related peri-muscular adipose tissue accelerates muscle atrophy.

Sarcopenia due to loss of skeletal muscle mass and strength leads to physical inactivity and decreased quality of life. The number of individuals with sarcopenia is rapidly increasing as the number of older people increases worldwide, making this condition a medical and social problem. Some patients with sarcopenia exhibit accumulation of peri-muscular adipose tissue (PMAT) as ectopic fat deposition surrounding atrophied muscle. However, an association of PMAT with muscle atrophy has not been demonstrated. Here, we show that PMAT is associated with muscle atrophy in aged mice and that atrophy severity increases in parallel with cumulative doses of PMAT. We observed severe muscle atrophy in two different obese model mice harboring significant PMAT relative to respective control non-obese mice. We also report that denervation-induced muscle atrophy was accelerated in non-obese young mice transplanted around skeletal muscle with obese adipose tissue relative to controls transplanted with non-obese adipose tissue. Notably, transplantation of obese adipose tissue into peri-muscular regions increased nuclear translocation of FoxO transcription factors and upregulated expression FoxO targets associated with proteolysis (Atrogin1 and MuRF1) and cellular senescence (p19 and p21) in muscle. Conversely, in obese mice, PMAT removal attenuated denervation-induced muscle atrophy and suppressed upregulation of genes related to proteolysis and cellular senescence in muscle. We conclude that PMAT accumulation accelerates age- and obesity-induced muscle atrophy by increasing proteolysis and cellular senescence in muscle.

A novel polysaccharide derived from algae extract inhibits cancer progression via JNK, not via the p38 MAPK signaling pathway.

Cancer has long been one of the most malignant diseases worldwide. Processes in cancer cells are often mediated by Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and other signaling pathways. Traditional therapies are often problematic. Recently, a novel polysaccharide derived from algae extract was investigated due to the increasing interest in biological activities of compounds from marine organisms. The effect of this novel polysaccharide on human MKN45 gastric carcinoma cells was determined previously. The current aimed to determine whether the polysaccharide affects other types of cancer, and the deeper mechanisms involved in the process. Human MCF-7 breast cancer cells were used to investigate the novel polysaccharide for its role in the cell growth and migration, and determine the mechanisms affected. MTT assay, nuclear staining and fluorescence activated cell sorting analysis demonstrated that the novel polysaccharide reduced the viability of MCF-7 cells by inducing cell apoptosis and arresting the cells at G2/M phase. Results of western blot analysis demonstrated that phosphorylation of JNK and expression of p53, caspase-9 and caspase-3 were upregulated in the polysaccharide-treated MCF-7 cells. SP600125, an inhibitor of JNK, maintained MCF-7 cell viability, prevented cell apoptosis and cycle arrest, and downregulated the polysaccharide-induced protein phosphorylation/expression. However, a migration assay demonstrated that the novel polysaccharide did not change the migration of MCF-7 cells, as well as the expression of p38 MAPK, and matrix metalloproteinase-9 and -2. Taken together, the current study demonstrated that the novel polysaccharide suppressed cancer cell growth, induced cancer cell apoptosis and cell cycle arrest via JNK signaling, but had no effect on cancer cell migration and p38 MAPK signaling.

Age-dependent increase in angiopoietin-like protein 2 accelerates skeletal muscle loss in mice.

Skeletal muscle atrophy, or sarcopenia, is commonly observed in older individuals and in those with chronic disease and is associated with decreased quality of life. There is recent medical and broad concern that sarcopenia is rapidly increasing worldwide as populations age. At present, strength training is the only effective intervention for preventing sarcopenia development, but it is not known how this exercise regimen counteracts this condition. Here, we report that expression of the inflammatory mediator angiopoietin-like protein 2 (ANGPTL2) increases in skeletal muscle of aging mice. Moreover, in addition to exhibiting increased inflammation and accumulation of reactive oxygen species (ROS), denervated atrophic skeletal muscles in a mouse model of denervation-induced muscle atrophy had increased ANGPTL2 expression. Interestingly, mice with a skeletal myocyte-specific Angptl2 knockout had attenuated inflammation and ROS accumulation in denervated skeletal muscle, accompanied by increased satellite cell activity and inhibition of muscular atrophy compared with mice harboring wildtype Angptl2 Moreover, consistent with these phenotypes, wildtype mice undergoing exercise training displayed decreased ANGPTL2 expression in skeletal muscle. In conclusion, ANGPTL2 up-regulation in skeletal myocytes accelerates muscle atrophy, and exercise-induced attenuation of ANGPTL2 expression in those tissues may partially explain how exercise training prevents sarcopenia.

A novel polysaccharide derived from algae extract induces apoptosis and cell cycle arrest in human gastric carcinoma MKN45 cells via ROS/JNK signaling pathway.

In recent years, interest in biological activities of compounds from marine organisms has intensified. Cancer is the most principal enemy for human life and health. For the first time, to the best of our knowledge, we investigated a novel algae-derived polysaccharide for its role in inducing apoptosis and cell cycle arrest in human gastric carcinoma MKN45 cells. We found that the novel polysaccharide suppressed MKN45 cell proliferation, induced cell apoptosis and arrested the cells at G2/M phase. Furthermore, we observed that the generation of reactive oxygen species (ROS) and the phosphorylation of Jun N-terminal kinase (JNK), p53, caspase-9 and -3 were induced in the polysaccharide-treated MKN45 cells. In addition, pretreatment with N-acetyl-cysteine (NAC) and SP600125, the inhibitor of ROS and JNK, induced MKN45 cell proliferation, prevented the cell apoptosis and released the cells from cycle arrest. Finally, we found that pretreatment with NAC prevented the JNK, p53, caspase-9 and -3 protein phosphorylation induced by the polysaccharide, however, pretreatment with SP600125 did not affect the generation of ROS, suggesting that ROS is upstream of JNK. Taken together, the novel polysaccharide induced cancer cell apoptosis and arrested cell cycle via ROS/JNK signaling pathway.

Cdk5rap1-mediated 2-methylthio modification of mitochondrial tRNAs governs protein translation and contributes to myopathy in mice and humans.

Transfer RNAs (tRNAs) contain a wide variety of posttranscriptional modifications that are important for accurate decoding. Mammalian mitochondrial tRNAs (mt-tRNAs) are modified by nuclear-encoded tRNA-modifying enzymes; however, the physiological roles of these modifications remain largely unknown. In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. The KO mice were highly susceptive to stress-induced mitochondrial remodeling and exhibited accelerated myopathy and cardiac dysfunction under stressed conditions. Furthermore, we demonstrate that the ms(2) modifications of mt-tRNAs were sensitive to oxidative stress and were reduced in patients with mitochondrial disease. These findings highlight the fundamental role of ms(2) modifications of mt-tRNAs in mitochondrial protein synthesis and their pathological consequences in mitochondrial disease.

Targeting complement anaphylatoxin C5a receptor in hyperoxic lung injury in mice.

Receptor binding of complement anaphylatoxin C5a results in proinflammatory activation of numerous diseases, but the role of receptor-mediated action during hyperoxic lung injury has, to the best of our knowledge, not yet been investigated. The contribution of the C5a receptor (C5aR) to hyperoxic lung injury in mice was investigated in this study. The effect of C5aR on hyperoxic lung injury in Balb/c mice was examined employing a C5aR antagonist (C5aRA). The mice were ventilated with 100% oxygen for 36 h with or without the administration of C5aRA. C5aR expression levels in non­treated or 100% oxygen-treated mice were assessed by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. The body weight and the relative lung weight of the mice, and the morphological changes in the lung were then determined. The total cell counts and the number of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) were determined using cytocentrifuge slides and a hemocytometer. The levels of interleukin-6 (IL-6), monocyte chemotactic protein (MCP-1) and tumor necrosis factor-α (TNF-α) in BALF and the myeloperoxidase (MPO) activity in the lung tissue were measured by enzyme-linked immunosorbent assay. The relative levels of CD68 and F4/80 messenger ribonucleic acid (mRNA) expression in the lung tissue were detected by RT-PCR. The TNF-α, IL-6 and MCP-1 protein expression levels in the lung tissue were assessed by western blot analysis. The results revealed hyperoxia-induced morphological changes, lung injury and increased expression levels of C5aR in lung tissue. The hyperoxia-induced increases in the total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were all significantly reduced in the mice receiving C5aRA. Treatment with C5aRA also attenuated the morphological changes and reduced MPO activity, and CD68 and F4/80 mRNA expression levels in the lung tissue, as well as the levels of IL-6, MCP-1 and TNF-α in BALF and lung tissue. In conclusion, C5a-C5aR action accelerated hyperoxia-induced lung injury, but this hyperoxic lung injury was attenuated by treatment with C5aRA.

Quantitative PCR measurement of tRNA 2-methylthio modification for assessing type 2 diabetes risk.

BACKGROUND: Genetic variants in the human CDKAL1 (CDK5 regulatory subunit associated protein 1-like 1) gene have been associated with reduced insulin secretion and type 2 diabetes (T2D). CDKAL1 is a methylthiotransferase that catalyzes 2-methylthio (ms(2)) modification of the adenine at position 37 (A37) of cytoplasmic tRNA(Lys)(UUU). We investigated the ms(2)-modification level of tRNA(Lys)(UUU) as a direct readout of CDKAL1 enzyme activity in human samples. METHOD: We developed a quantitative PCR (qPCR)-based method to measure ms(2) modification. tRNA(Lys)(UUU) was reverse-transcribed with 2 unique primers: Reverse primer r1 was designed to anneal to the middle of this tRNA, including the nucleotide at A37, and reverse primer r2 was designed to anneal to the region downstream (3') of A37. Subsequent qPCR was performed to detect the corresponding transcribed cDNAs. RESULTS: The efficiency of reverse transcription of tRNA(Lys)(UUU) was ms(2)-modification dependent. The relative difference in threshold cycle number obtained with the r1 or r2 primer yielded the ms(2)-modification level in tRNA(Lys)(UUU) precisely as predicted by an original mathematical model. The method was capable of measuring ms(2)-modification levels in tRNA(Lys)(UUU) in total RNA isolated from human peripheral blood samples, revealing that the ms(2)-modification rate in tRNA(Lys)(UUU) was decreased in individuals carrying the CDKAL1 genotype associated with T2D. In addition, the ms(2)-modification level was correlated with insulin secretion. CONCLUSIONS: The results point to the critical role of ms(2) modification in T2D and to a potential clinical use of a simple and high-throughput method for assessing T2D risk.

A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro.

Diabetic retinopathy is a common complication of diabetes mellitus (DM). The oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose closely approximates the molecular basis for the loss of vision associated with this disease. We investigate a novel algae-derived polysaccharide compound for its role in protecting ARPE-19 cells from high glucose-induced oxidative damage. ARPE-19 cells were cultured for 4 d with normal concentration of D-glucose, and exposed to either normal or high concentrations of D-glucose in the presence or absence of the polysaccharide compound at variety of concentrations for another 48 h. Taurine was used as a positive control. Activity of super oxide dismutase (SOD) and concentration of glutathione (GSH) were measured as well as cytotoxicity of high glucose and the polysaccharide compound. To analyse cellular damage by high glucose, activation of Annexin V and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) were examined. Our results showed that a significant cellular damage on ARPE-19 cells after 48 h treatment with high glucose, accompanied by a decrease in SOD activity and GSH concentration; high glucose also caused ARPE-19 cell apoptosis and activation of p38MAPK and ERK. As the non-toxic polysaccharide compound protected ARPE-19 cells from high glucose-induced cellular damage, the compound recovered SOD activity and concentration of GSH in the cells. The compound also abrogated the cell apoptosis and activation of p38MAPK and ERK. Therefore, the polysaccharide compound derived from algae extracts could be unique candidate for a new class of anti-DM and anti-oxidative damage.

Inhibitory effects of C4a on chemoattractant and secretagogue functions of the other anaphylatoxins via Gi protein-adenylyl cyclase inhibition pathway in mast cells.

A recombinant complement anaphylatoxin, C4a, inhibited chemotaxis, respiratory burst and histamine release in mast cell-like HMC-1 cells that were treated with recombinant C5a anaphylatoxin. C4a also inhibited histamine release from HMC-1 cells that were induced by recombinant C3a. The inhibition of C5a- and C3a-induced leukocyte reactions by C4a was recapitulated in peripheral blood CD133(+) cell-derived differentiated mast cells. In HMC-1 cells, C4a inhibited cytoplasmic Ca(2+) influx, an event that precedes anaphylatoxin-induced chemotactic and secretary responses. A conditioned medium of HMC-1 cells after shortly treated with C4a also inhibited the anaphylatoxin-induced Ca(2+) influx even after removal of C4a, indicating that the effect of C4a is to liberate an autocrine inhibitor from the mast cells. The inhibitor secretion by C4a was prevented with pertussis toxin or with a phosphodiesterase inhibitor. Conversely, an adenylyl cyclase inhibitor reproduced the effect of C4a. C4a decreased the intracellular cyclic AMP concentration of HMC-1 cells, indicating that C4a elicited the Gi protein-adenylyl cyclase inhibition pathway. Neither C4a nor the conditioned medium, however, inhibited Ca(2+) influx and respiratory burst in C5a- or C3a-stimulated peripheral neutrophils, suggesting that these cells lack this inhibitory system. Additionally, in HMC-1 cells, C4a did not inhibit Ca(2+)-independent, Leu72Gln-C5a-stimulated chemotactic response. In agreement with this finding, C4a treatment inhibited ERK1/2 phosphorylation in HMC-1 cells stimulated with other anaphylatoxins but did not inhibit p38MAPK phosphorylation in cells stimulated with Leu72Gln-C5a. Taken together, these findings suggest that the autocrine inhibitory effect elicited by C4a is attributed to interruption of Ca(2+)-dependent intracellular signaling pathway.