Intracellular Free Amino Acid Research Articles

Insights into the complementation potential of the extreme acidophile's orthologue in replacing Escherichia coli hfq gene-particularly in bacterial resistance to environmental stress.

Acidithiobacillus caldus is a typical extreme acidophile widely used in the biohydrometallurgical industry, which often experiences extreme environmental stress in its natural habitat. Hfq, an RNA-binding protein, typically functions as a global regulator involved in various cellular physiological processes. Yet, the biological functions of Hfq derived from such extreme acidophile have not been extensively investigated. In this study, the recombinant strain Δhfq/Achfq, constructed by CRISPR/Cas9-mediated chromosome integration, fully or partially restored the phenotypic defects caused by hfq deletion in Escherichia coli, including impaired growth performance, abnormal cell morphology, impaired swarming motility, decreased stress resistance, decreased intracellular ATP and free amino acid levels, and attenuated biofilm formation. Particularly noteworthy, the intracellular ATP level and biofilm production of the recombinant strain were increased by 12.2% and 7.0%, respectively, compared to the Δhfq mutant. Transcriptomic analysis revealed that even under heterologous expression, AcHfq exerted global regulatory effects on multiple cellular processes, including metabolism, environmental signal processing, and motility. Finally, we established a potential working model to illustrate the regulatory mechanism of AcHfq in bacterial resistance to environmental stress.

Effects of the Toxic Non-Protein Amino Acid β-Methylamino-L-Alanine (BMAA) on Intracellular Amino Acid Levels in Neuroblastoma Cells.

The cyanobacterial non-protein amino acid (AA) β-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.

Autophagy contributes to increase the content of intracellular free amino acids in hard clam (Mercenaria mercenaria) during prolonged exposure to hypersaline environments

Marine bivalves in intertidal zones and land-based seawater ponds are constantly subjected to a wide range of salinity fluctuations due to heavy rainfall, intense drought, and human activities. As osmoconformers, bivalves rely primarily on rapid release or accumulation of free amino acids (FAAs) for osmoregulation. Euryhaline bivalves are capable of withstanding hyposaline and hypersaline environments through regulation of physiology, metabolism, and gene expression. However, current understanding of the molecular mechanisms underlying osmoregulation and salinity adaptation in euryhaline bivalves remains largely limited. In this study, RNA-seq, WGCNA and flow cytometric analysis were performed to investigate the physiological responses of hard clams (Mercenaria mercenaria) to acute short-term hyposalinity (AL) and hypersalinity (AH), and chronic long-term hyposalinity (CL) and hypersalinity (CH) stress. We found that amino acids biosynthesis was significantly inhibited and aminoacyl-tRNA biosynthesis was augmented to decrease intracellular osmolarity during hyposaline exposure. Under CH, numerous autophagy-related genes (ATGs) were highly expressed, and the autophagy activity of gill cells were significantly up-regulated. A significant decrease in total FAAs content was observed in gills after NH4Cl treatment, indicating that autophagy was crucial for osmoregulation in hard clams during prolonged exposure to hypersaline environments. To prevent premature or unnecessary apoptosis, the expression of cathepsin L was inhibited under AL and AH, and inhibitors of apoptosis was augmented under CL and CH. Additionally, neuroendocrine regulation was involved in salinity adaption in hard clams. This study provides novel insights into the physiological responses of euryhaline marine bivalves to hyposaline and hypersaline environments.

Can hyper/hypo-osmoregulating fiddler crabs from the Atlantic coast of South America mobilize intracellular free amino acids as osmotic effectors during salinity challenge?

Weakly osmoregulating crustaceans use intracellular free amino acids (FAA) to attenuate cell volume changes consequent to alterations in hemolymph osmolality. Whether semiterrestrial, strong hyper/hypo-osmoregulators exhibit this ability is unknown. We investigate FAA mobilization in muscle tissue of 10 fiddler crabs from the genera Minuca, Leptuca, and Uca distributed along the Atlantic coast of South America. Crabs were subjected to severe hypo- or hyper-osmotic challenge at their lower or upper critical salinity limits for 5 days; reference crabs were held in isosmotic media. Hemolymph osmolality was measured, chela muscle FAA were identified and quantified, and percent contribution to intracellular osmolality (%FAA) was calculated. At isosmoticity, total FAA were nominally twofold higher in Minuca species (≈116 mmol/kg wet mass) compared to Uca (≈60 mmol/kg wet mass). Glycine, alanine, arginine, and taurine constituted >80% of the total FAA pool. On hyperosmotic challenge, hemolymph osmolalities ranged from 843 to 1282 mOsm/kg H2 O. FAA increased, although %FAA remained unaltered. Hypo-osmoregulating crabs thus can mobilize FAA, likely owing to a lesser ability to secrete salt near their upper critical limits. On hypo-osmotic challenge, osmolalities were regulated more tightly, between 475 and 736 mOsm/kg H2 O. Total FAA and %FAA showed little change, probably due to the crabs' strong hyper-osmotic extracellular regulatory ability, FAA consequently playing a diminished role in isosmotic intracellular regulation (IIR). Total FAA responses to hyper/hypo-osmotic challenge are thus asymmetrical. The lack of phylogenetic signal in FAA mobilization suggests that closely related fiddler crabs do not share similar strategies of IIR.

Mechanism of taurine reducing inflammation and organ injury in sepsis mice

The fundamental basis for the pathogenesis of sepsis is an inflammatory imbalance, which is considered to be the main target for treatment. Taurine is an intracellular free amino acid that has anti-inflammatory and antioxidant effects. To investigate the protective mechanism of taurine in sepsis, we used in vitro and in vivo experiments to explore the effects of taurine on neutrophil and monocyte immune function. Metabolomic analysis showed large amounts of taurine in neutrophils and monocytes and a dramatic decrease in taurine levels after LPS exposure. Taurine supplementation decreased the expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) in LPS-challenged neutrophils and monocytes and reduced the formation of neutrophil extracellular traps by restricting reactive oxygen species. Moreover, taurine protected septic mice from death, improved tissue injuries in the lung, liver, and kidney by reducing neutrophil infiltration and TNF-α production. Our data indicate that a supplement with taurine might be a promising therapeutic strategy for sepsis to reduce hyper inflammation and improve multi-organ dysfunctions.

Metabolic Effects of Cedar Tar on Colon Cancer Cell Line (HCT-116): A Follow-up Study

Objective: Colon cancer (CC) is considered to be the most common cancers seen worldwide. Surgery and chemotherapy are the leading methods used in treatment. In recent years, the therapeutic properties of natural products have been investigated and used in drug development. Cedrus libani plant that normally grows in Syria and Turkey, some studies have shown that the plant’s extract (cedar tar) has anti-microbial and anti-cancer properties on colon cancer. Recently, metabolic changes have been identified as the hallmark of cancer, and various metabolic drugs are being investigated in clinical trials. This study was carried out to investigate the effects of cedar tar on the amino acid metabolism of colon cancer cells. Materials and Methods: Colon cancer cells (HCT-116) were homogenized in cold PBS medium after incubation with 30 µg/mL dose of cedar tar for 24 hours in appropriate medium. The intracellular free amino acid profile in the samples was analyzed by LC-MS/MS method. Statistical analysis of the obtained data was done with SPSS and MetaboAnalyst 5.0 program. Results: According to the statistical analysis results, significant positive correlations were found between β-alanine, anserine amino acids and apoptotic effects of cedar tar between the untreated control and cedar tar-treated groups, while a negative correlation was found with O-Phosphorylethanolamine amino acid. Conclusion: By increasing the levels of β-alanine and anserine amino acids in colon cancer cells, cedar tar reduces both the metastatic character of the cells and inhibits tumor formation; It caused cell proliferation and energy deprivation by reducing the level of O-Phosphorylethanolamine amino acid, which is defined as a pro-tumorigenic metabolite. Cedar tar both triggered apoptosis and changed amino acid metabolism in colon cancer cells. It has been determined that cedar tar has the potential to be used as a natural product in the treatment of colon cancer.

The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.

Protective effect of taurine on sepsis‑induced lung injury via inhibiting the p38/MAPK signaling pathway

Sepsis, a leading cause of acute lung injury (ALI), is characterized by an overwhelming systemic inflammatory response and widespread organ injury, particularly in the lungs. Taurine, an intracellular free amino acid, has been used for the treatment of various diseases, including lung injury; however, the underlying mechanisms are unclear. The present study aimed to investigate the protective effect of taurine on septic ALI and the underlying mechanism. A septic ALI model was established by performing cecal ligation and puncture (CLP) surgery on Sprague Dawley rats. Following successful model establishment, rats were treated with taurine. The results of hematoxylin and eosin, respiratory function detection, malondialdehyde level and superoxide dismutase activity determination and ELSIA demonstrated that taurine significantly alleviated lung injury, restored respiratory function, reduced oxidation and decreased the concentrations of inflammatory factors in CLP-induced septic ALI model rats. In addition, compared with that in the ALI group, western blotting results indicated that taurine ameliorated lung epithelial injury by significantly increasing the expression levels of lung epithelial markers, E-cadherin and occludin. The western blotting results demonstrated that, compared with the control group, the p38/MAPK and NF-κB signaling pathways were significantly activated in CLP-induced septic ALI model rats, but taurine significantly suppressed ALI-mediated signaling pathway activation. To investigate the mechanism underlying taurine in the treatment of septic ALI, CLP-induced septic ALI model rats were treated with an antagonist of the p38/MAPK signaling pathway (SB203580). The effects of SB203580 on CLP-induced septic ALI model rats were similar to those of taurine. SB203580 significantly attenuated sepsis-induced lung injury and increases in IL-1β and TNF-α concentrations in the lung tissue. In addition, SB203580 promoted restoration of the injured lung tissue and respiratory function in CLP-induced septic ALI model rats. The western blotting results indicated that SB203580 significantly decreased the ratios of phosphorylated (p)-p38/p38 and p-p65/065, and increased the protein expression levels of E-cadherin and occludin compared with those in the ALI group. In summary, the present study demonstrated that taurine alleviated sepsis-induced lung injury, which was associated with suppression of the inflammatory response and oxidative stress via inhibiting the p38/MAPK signaling pathway. Therefore, the p38/MAPK signaling pathway may serve as a potential therapeutic target for the treatment of sepsis-induced ALI.

CCT3 suppression prompts apoptotic machinery through oxidative stress and energy deprivation in breast and prostate cancers

Mediated by chaperon proteins, protein misfolding plays a crucial role in cancer pathogenesis. Chaperonin Containing TCP1 Subunit 3 (CCT3) is one of eight subunits forming eukaryotic chaperons that catalyzes correct folding of the proteins employed in cell division, proliferation, and apoptosis pathway. Moreover, CCT3 expression increases responsively with carcinogenesis. However, how CCT3 drives the cancerous process has not been documented. Here we probed the mechanistic and functional interactions between CCT3 and apoptotic pathways and cell stressors. First, we profiled CCT3 expression levels of different 16 cell lines and found that CCT3 expression levels of CRL-2329 and PC3 were significantly increased. Then, we suppressed CCT3 levels in CRL-2329 and PC3 lines by miR-24–3p, miR-128–3p, and miR-149–5p mimics, and measured apoptotic response of the cell lines to the knockdown of CCT3 by acridine orange/ethidium bromide and Annexin V/PI staining, cell-cycle and mitochondria membrane potential (MMP) analyses, intracellular reactive oxygen species (ROS) measurement and analysis of expression levels of the apoptotic genes. After having suppressed CCT3, the cell cycle was arrested in the G0/G1 phase, MMP was impaired, and the intracellular ROS level was increased. These signs of apoptotic flux were corroborated by morphological images, statistically enhanced expression levels of the apoptotic pathway modulators and intracellular free amino acids profile. The free amino acid profile, which is heavily implicated in energy metabolism and cell division, is fluctuated in the progress of canceration. Strikingly, suppressed CCT3 shifted intracellular levels of glutamine, beta-alanine, glycine, serin, asparagine and sarcosine, which are employed in energy metabolism. Consequently, miRNA-mediated CCT3 suppression spur apoptosis by unbalancing the homeostasis in intracellular ROS and the profile of free amino acids in energy metabolism. Taken together, we anticipate that miRNA-mediated CCT3 suppression might provide a “dual therapeutic strategy” through conventional cellular toxicity as well as energy withdrawal.

Comparison of Amino Acid Metabolisms in Normal Prostate (PNT-1A) and Cancer Cells (PC-3)

Prostate cancer is the second most common cancer in men. Prostate-specific antigen (PSA) levels, commonly used in the diagnosis of prostate cancer, are increased in both malign and benign conditions, such as prostate hyperplasia (BPH) and prostatitis. Thus, more specific markers are urgently needed to discriminate between prostate cancer and benign diseases of the prostate. The purpose of this study is to examine both the intracellular and extracellular free amino acid profiles of metastatic prostate cancer cells (PC-3), normal prostate cells (PNT-1A), and metabolic changes (e.g., pH). In this study, cancer and normal cells were incubated in the appropriate medium. Then, the cells were collected and lysed in a cold medium. Finally, intracellular and extracellular free amino acid profiles were analyzed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Extracellular metabolites were analyzed via blood gas measurements, including pH, CO₂, and O₂. We determined that intracellular prostate cancer cells include high amounts of glutamic acid, aspartic acid, glutamine, alpha aminobutyric acid, glycine, and proline, while cancer cells mostly use hydroxyproline, serine, alpha aminobutyric acid, ethanolamine, and valine from the extracellular medium. It was also determined that the extracellular pH of cancer cells is more acidic than that of normal cells, and normal cells have higher levels of Ca⁺, Cl^-, and Glu. In this study, we found that group amino acids have significant potential in prostate cancer pathogenesis; therefore, they have potential usefulness as a biomarker in the diagnosis of prostate cancer.

Abstract PR22: Collagen scavenging alters macrophage phenotype in pancreatic cancer

Abstract A hallmark of pancreatic tumors is their highly desmoplastic stroma composed of fibroblasts, immune cells and a dense network of collagen fibers. Pancreatic tumor-associated macrophages are considered M2 macrophages and are thought to support tumor progression through the engagement of multiple mechanisms that promote immune tolerance. Another well-documented role of M2 macrophages is the remodeling of the tumor stroma via collagen production and degradation, with the latter being mediated by mannose receptor (Mrc1)-mediated endocytosis of collagen. Given our previous work showing that protein scavenging by tumor cells can be utilized as a nutrient delivery mechanism, we set out to investigate the functional significance of collagen scavenging by macrophages. We have found that, in M2 macrophages, Mrc1-mediated collagen internalization and subsequent lysosomal degradation leads to a metabolic reprogramming that is characterized by the accumulation of collagen-derived intracellular free amino acids and an increase in arginine biosynthesis. The increase in arginine levels results in a significant upregulation of inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1), giving rise to an M1/M2 hybrid macrophage. Moreover, iNOS+ARG1+ double-positive macrophages are present in the microenvironment of pancreatic tumors. The functional contribution of this cell population to pancreatic tumorigenesis is being investigated. Our findings suggest a new modality for the functional modulation of tumor-associated macrophages through the coupling of extracellular matrix remodeling to protein scavenging. This abstract is also being presented as Poster A58. Citation Format: Madeleine M. LaRue, Jane Cullis, Dafna Bar-Sagi. Collagen scavenging alters macrophage phenotype in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR22.

Metabolomic analysis of Shiga toxin 2a-induced injury in conditionally immortalized glomerular endothelial cells.

Shiga toxin 2a (Stx2a) induces hemolytic uremic syndrome (STEC HUS) by targeting glomerular endothelial cells (GEC). We investigated in a metabolomic analysis the response of a conditionally immortalized, stable glomerular endothelial cell line (ciGEnC) to Stx2a stimulation as a cell culture model for STEC HUS. CiGEnC were treated with tumor necrosis factor-(TNF)α, Stx2a or sequentially with TNFα and Stx2a. We performed a metabolomic high-throughput screening by lipid- or gas chromatography and subsequent mass spectrometry. Metabolite fold changes in stimulated ciGEnC compared to untreated cells were calculated. 320 metabolites were identified and investigated. In response to TNFα + Stx2a, there was a predominant increase in intracellular free fatty acids and amino acids. Furthermore, lipid- and protein derived pro-inflammatory mediators, oxidative stress and an augmented intracellular energy turnover were increased in ciGEnC. Levels of most biochemicals related to carbohydrate metabolism remained unchanged. Stimulation of ciGEnC with TNFα + Stx2a is associated with profound metabolic changes indicative of increased inflammation, oxidative stress and energy turnover.

Effects of varying extracellular amino acid profile on intracellular free amino acid concentrations and cell signaling in primary mammary epithelial cells

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, -F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.

The Autophagic Flux Inhibitor Bafilomycine A1 Affects the Expression of Intermediary Metabolism-Related Genes in Trout Hepatocytes.

Autophagy is an evolutionarily conserved process of cellular self-eating which emerged these last years as a major adaptive metabolic response to various stresses such as fasting, hypoxia, or environmental pollutants. However, surprisingly very few data is currently available on its role in fish species which are directly exposed to frequent environmental perturbations. Here, we report that the treatment of fasted trout hepatocytes with the autophagy inhibitor Bafilomycine A1 lowered the mRNA levels of many of the gluconeogenesis-related genes and increased those of genes involved in intracellular lipid stores. Concurrently, intracellular free amino acid levels dropped and the expression of the main genes involved in the endoplasmic reticulum (ER) stress exhibited a sharp increase in autophagy inhibited cells. Together these results highlight the strong complexity of the crosstalk between ER, autophagy and metabolism and support the importance of considering this function in future studies on metabolic adaptation of fish to environmental stresses.

Effects of amino acid availability on the intracellular free amino acid concentration

Effects of amino acid availability on the intracellular free amino acid concentration

Numerical analysis of intracellular amino acid profiles of breast cancer cells with K-Ras or PI3K mutation in response to kinase inhibitors

BackgroundVarious efforts to understand the relationship between biological information and disease have been done using many different types of highthroughput data such as genomics and metabolomics. However, information obtained from previous studies was not satisfactory, implying that new direction of studies is in need. Thus, we have tried profiling intracellular free amino acids in normal and cancerous cells to extract some information about such relationship by way of the change in IFAA levels in response to the treatment of three kinase inhibitors. We define two measures such as relative susceptibility (RS) and relative efficacy (RE) to numerically quantify susceptibility of cell line to treatment and efficacy of treatment on cell line, respectively.MethodsWe applied principal component analysis (PCA) to the intracellular free amino acids (IFAAs) of isogenic breast cells with oncogenic mutation in K-Ras or PI3K genes to investigate the change in IFAA levels in response to the treatment of three kinase inhibitors. Two-dimensional plot, which was graphically represented by using the first two principal components (PCs), enabled us to evaluate the treatment efficacy in cancerous cells in terms of the quantitative distance of two IFAA profiles from cancerous and normal cells with the same treatment condition.ResultsThe biggest change in metabolic states in K-Ras mutant cell was caused by REGO for both treatment time (RS=2.31 (24 h) and 1.64 (48 h)). Regarding RE, REGO was the most effective on K-Ras/PI3K mutant cell line for treatment time 24h (RE=1.28) while PI3K inhibitor had good effect on K-Ras mutant cell line for 48h (RE=1.1).ConclusionsNumerical study on the link between amino acid profile and cancer has been done in two different dimensions. We then summarized such link in terms of two new metrics such as RS and RE, which we first define in this work. Although our study based on those metrics seems to work, we think that the usefulness of the metrics in cancer study of this kind need to be further investigated.

Taurine enhances the protective effect of Dexmedetomidine on sepsis-induced acute lung injury via balancing the immunological system

Sepsis, an overwhelming systemic inflammatory disease, is the leading cause of acute lung injury (ALI). Despite plenty of researches have been done, effective drugs treating septic ALI are still eagerly needed in the clinic. Dexmedetomidine (Dex), a potent alpha-2 adrenoreceptor agonist, has been reported to possess antioxidant, anti-apoptosis and anti-inflammatory abilities. Taurine, a kind of intracellular free amino acid, has been used to treat various diseases. This study aimed to explore the combination effect of Dex and Taurine on septic ALI and the underlying mechanism in vivo. The establishment of septic ALI was set up in SD rats by cecal ligation and puncture (CLP) operation. Results indicated that Dex or Taurine could reduce septic ALI-induced cell apoptosis via decreasing caspase-3 activity. However, the combination of Dex or Taurine produced greater effect. Besides that, Dex combined with Taurine could better promote cell proliferation with remarkably elevated Ki67 expression. The combination of Dex and Taurine significantly suppressed the activation of NF-κB pathway via inhibiting P65 phosphorylation and P65 nuclear translocation, leading to the down-regulation of interleukin (IL)-6 and IL-1β. Moreover, co-administration of Dex and Taurine alleviated the imbalance of Th1/Th2 induced by septic ALI to a great extent. All in all, our study suggested the synergistic therapeutic effect of Dex and Taurine on septic ALI.

The Mitotic and Metabolic Effects of Phosphatidic Acid in the Primary Muscle Cells of Turbot (Scophthalmus maximus).

Searching for nutraceuticals and understanding the underlying mechanism that promote fish growth is at high demand for aquaculture industry. In this study, the modulatory effects of soy phosphatidic acids (PA) on cell proliferation, nutrient sensing, and metabolic pathways were systematically examined in primary muscle cells of turbot (Scophthalmus maximus). PA was found to stimulate cell proliferation and promote G1/S phase transition through activation of target of rapamycin signaling pathway. The expression of myogenic regulatory factors, including myoD and follistatin, was upregulated, while that of myogenin and myostatin was downregulated by PA. Furthermore, PA increased intracellular free amino acid levels and enhanced protein synthesis, lipogenesis, and glycolysis, while suppressed amino acid degradation and lipolysis. PA also was found to increased cellular energy production through stimulated tricarboxylic acid cycle and oxidative phosphorylation. Our results identified PA as a potential nutraceutical that stimulates muscle cell proliferation and anabolism in fish.

Taurine Administration Recovers Motor and Learning Deficits in an Angelman Syndrome Mouse Model

Angelman syndrome (AS, MIM 105830) is a rare neurodevelopmental disorder affecting 1:10–20,000 children. Patients show moderate to severe intellectual disability, ataxia and absence of speech. Studies on both post-mortem AS human brains and mouse models revealed dysfunctions in the extra synaptic gamma-aminobutyric acid (GABA) receptors implicated in the pathogenesis. Taurine is a free intracellular sulfur-containing amino acid, abundant in brain, considered an inhibiting neurotransmitter with neuroprotective properties. As taurine acts as an agonist of GABA-A receptors, we aimed at investigating whether it might ameliorate AS symptoms. Since mice weaning, we orally administered 1 g/kg/day taurine in water to Ube3a-deficient mice. To test the improvement of motor and cognitive skills, Rotarod, Novel Object Recognition and Open Field tests were assayed at 7, 14, 21 and 30 weeks, while biochemical tests and amino acid dosages were carried out, respectively, by Western-blot and high-performance liquid chromatography (HPLC) on frozen whole brains. Treatment of Ube3am−/p+ mice with taurine significantly improved motor and learning skills and restored the levels of the post-synaptic PSD-95 and pERK1/2-ERK1/2 ratio to wild type values. No side effects of taurine were observed. Our study indicates taurine administration as a potential therapy to ameliorate motor deficits and learning difficulties in AS.

Doxorubicin chemotherapy affects the intracellular and interstitial free amino acid pools in skeletal muscle.

Skeletal muscle (SM) health and integrity is dependent on the dynamic balance between protein synthesis and degradation, and central to this process is the availability of amino acids (AA) in the amino pool. While Doxorubicin (DOX) remains one of the most widely used chemotherapeutic agents for the treatment of solid and hematological malignancies, little is known of the effect of the drug on SM, particularly its effect on the availability of amino acids in the tissue. The purpose of this study was to examine the effect of DOX administration on vascular, interstitial and intracellular concentrations of AA in SM of the rat up to 8 days after the administration of a 1.5 or 4.5 mg/kg i.p. dose of DOX. In the plasma, total amino acids (TAA) were significantly increased compared to control where greater (P<0.05) concentrations were observed following the 1.5 mg/kg dose compared to the 4.5 mg/kg dose. Compared to control, the 1.5 mg/kg dose resulted in an increase (P<0.05) in interstitial TAA whereas the 4.5 mg/kg resulted in a sustained decrease (P<0.05). Intracellular TAA, essential amino acids (EAA) and branched-chain amino acids (BCAA) where significantly increased in each muscle group analyzed, following the 1.5 and 4.5 mg/kg doses compared to control. This study provides important insight into the amino acid response following DOX chemotherapy and presents a substantial foundation for future studies focused on reducing SM damage and recovery by targeting amino acid metabolism.