Uncovering the mechanisms of MuRF1-induced ubiquitylation and revealing similarities with MuRF2 and MuRF3.

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene) type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with a UBE2 (ubiquitin conjugating enzyme). Our understanding of MuRF1 function remains unclear as candidate UBE2s have not been fully elucidated. In the present study, we screened human ubiquitin dependent UBE2s in vitro and found that MuRF1 engages in ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. MuRF1 can cause mono-ubiquitylation, K48- and K63-linked polyubiquitin chains in a UBE2 dependent manner. Moreover, we identified a two-step UBE2 dependent mechanism whereby MuRF1 is monoubiquitylated by UBE2W which acts as an anchor for UBE2N/V to generate polyubiquitin chains. With the in vitro ubiquitylation assay, we also found that MuRF2 and MuRF3 not only share the same UBE2 partners as MuRF1 but can also directly ubiquitylate the same substrates: Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). In summary, our work presents new insights into the mechanisms that underpin MuRF1 activity and reveals overlap in MuRF-induced ubiquitylation which could explain their partial redundancy in vivo.

Ubiquitin E3 ligase Atrogin-1 protein is regulated via the rapamycin-sensitive mTOR-S6K1 signaling pathway in C2C12 muscle cells.

Atrogin-1 and Muscle-specific RING finger protein 1 (MuRF1) are highly expressed in multiple conditions of skeletal muscle atrophy. The phosphoinositide 3-kinase (PI3K)/Akt/forkhead box (FoxO) signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mechanistic target of rapamycin complex 1 (mTORC1), which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 with a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 µM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased by PF-4708671 treatment for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by the rapamycin-sensitive mTOR-S6K1-dependent signaling pathway.

Antibacterial, anti-inflammatory and antioxidant activities of Mahanintangtong and its constituent herbs, a formula used in Thai traditional medicine for treating pharyngitis.

BACKGROUND: Mahanintangtong is listed in the Thailand's National List of Essential Medicines (NLEM). It is used to treat non-specific fevers and illnesses such as pharyngitis and chickenpox. In this study, we investigated the biological activities of the different medicinal plants used in the Mahanintangtong formula. METHODS: The plant materials were extracted by maceration and decoction. Antimicrobial activity, assessed by disc diffusion method, the minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were compared with commercially available standard antibiotics. To elucidate the anti-inflammatory mechanisms, inhibition of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) production was tested by Griess and ELISA techniques. Antioxidant activity was measured by ABTS and DPPH scavenging assays. RESULTS: The extracts with the best antimicrobial activities were carbonized Tectona grandis showing against Streptococcus pyogenes, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. The ethanol extract of Dracaena loureiroi wood exhibited the highest NO and IL-6 inhibitory activity with IC50 values of 9.42 ± 1.81 and 12.02 ± 0.30 µg/mL, respectively. The ethanol extract of Pogostemon cablin had the highest TNF-α inhibitory with IC50 values of 10.68 ± 0.02 µg/mL. In anti-free radical testing, the ethanol extract of D. loureiroi displayed high antioxidant activity by both ABTS and DPPH assays. CONCLUSION: The ethanol extracts from carbonized T. grandis and Mahanintangtong showed good antimicrobial activity, especially against S. pyogenes, and good anti-inflammatory activity. These findings are relevant to the pathogenesis of pharyngitis and justify additional studies to see if Mahanintangtong could have clinical utility.

Antioxidant activities and phytochemical constituents of Antidesma thwaitesianum Müll. Arg. leaf extracts.

OBJECTIVE: To investigate the antioxidant activities as well as phytochemical constituents of Antidesma thwaitesianum Müll. Arg. leaf extracts. METHODS: The leaves of A. thwaitesianum were extracted using three different methods: blending with distilled water, maceration with ethanol and decoction. The chemical antioxidant activity of the plant leaf extracts was evaluated using 2,2-diphenyl-1-picryhydrazyl (DPPH) radical and 2,2'-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS⁺) radical scavenging assays, as well as the ferric reducing antioxidant power assay. Cellular antioxidant activity was determined by superoxide and nitric oxide scavenging assays. The cytotoxicity of the leaf extracts in RAW 264.7 and differentiated HL-60 cells was tested in parallel using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays, respectively. The total phenolic and flavonoid contents were also assessed by spectrophotometric analysis. Phytochemical constituents of the most potent extract were investigated by liquid chromatography with an electrospray ionization quadrupole time-of-flight mass spectrometer (LC-ESI-QTOF-MS/MS). RESULTS: The ethanolic (ME) and decoction (LW) extracts of dried leaves had the highest chemical scavenging activity against DPPH and ABTS⁺ free radicals with half maximal effective concentration (EC50) values ranging from 3.54 to 6.44 µg/mL. ME and LW exerted moderate ferric reducing activity, with ferric reducing antioxidant power values of 847.41 and 941.26 mg Fe2+/g extract, respectively. Similarly, ME showed potent cellular scavenging activity against superoxide and nitric oxide radicals with EC50 values of 58.12 and 71.90 µg/mL, respectively. However, LW exhibited only strong nitric oxide scavenging activity with an EC50 value of 91.20 µg/mL. The cell viability of RAW 264.7 and HL-60 cells was greater than 70% in all tested concentrations of both extracts, thus confirming the absence of their cytotoxicity. ME and LW contained high total phenolic contents of 231.14 and 274.42 mg gallic acid equivalents per gram, respectively, as well as high total flavonoid contents of 18.82 and 22.17 mg quercetin equivalents per gram, respectively. LC-ESI-QTOF-MS/MS analysis revealed the presence of 52 structurally characterized compounds in ME, 43 of which were tentatively identified. Hydroxycinnamic acids such as caffeic acid and its derivatives were the predominant phenolic compounds. CONCLUSION: This is the first report describing potent chemical and cellular antioxidant effects of the ethanolic leaf extract of A. thwaitesianum. The extract contained high total phenolic and flavonoid contents. LC-ESI-QTOF-MS/MS analysis further revealed an abundance of caffeic acid derivatives and flavonoids. These data support its potential use as dietary supplements in oxidative stress prevention.

Cytotoxic, Anti-inflammatory and Antioxidant Activities of Heliotropium indicum Extracts.

Background: Heliotropium indicum Linn., or 'Indian heliotrope' is very common in India with a long history of traditional medicinal uses in many countries in the world. In Thailand, the plant has been traditionally use to cure various diseases such as fever, insect bites, stings, diarrhea, skin rashes, menstrual disorder and urticaria. In addition, the plant is commonly used by Thai folk doctors as a component in remedies for treatment of lung cancer. Objective: In the present study, we investigated cytotoxicity against two types of lung cancer cell lines (A549 and NCI-H226), anti-inflammatory effect and antioxidant activity of Heliotropium indicum extracts. Material and Method: The water and ethanolic extracts of Heliotropium indicum were tested. The cytotoxic activity against two types of human lung cancer cell lines (A549 and NCI-H226) was evaluated by sulforhodamine B (SRB) assay. The antiinflammatory effect was investigated on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. LPS-induced nitric oxide (NO) production was determined by Griess reagent. The antioxidant activity was performed by 1, 1-diphenyl-picrylhydrazyl (DPPH) radical scavenging method. Results: The ethanolic extract showed cytotoxic activity only against NCI-H226 (IC50 = 51.90±2.35 µg/ml) whereas the water extract had no cytotoxic activity against both A549 and NCI-H226 (IC50 >100 µg/ml). For anti-inflammatory effect, the results revealed that the ethanolic extract exhibited the most potent inhibitory activity on nitric oxide production (IC50 = 24.17±2.12 µg/ml), followed by Indomethacin (positive control) with an IC50 value of 34.67±6.23 µg/ml while water extract was apparently inactive (IC50 >100 µg/ml). For antioxidant activity, the ethanolic extract showed high antioxidant activity (EC50 = 28.91±4.26 µg/ml) but the water extract showed no antioxidant activity (EC50 >100 µg/ml). Conclusion: These results can support using Heliotropium indicum Linn. for component in lung cancer remedy by Thai folk doctors. However, more studies are required.

Antimicrobial, antioxidant activities and total phenolic content of Thai medicinal plants used to treat HIV patients.

BACKGROUND: Opportunistic infections in AIDs patients is the leading cause of death in among them. HIV infection was reported as causes of increasing oxidative stress which may lead to progress of many syndrome. Thus medicinal plants as demonstrated antimicrobial and antioxidant activities would be therapeutic values to treat opportunistic infections of AIDs patients. OBJECTIVE: To investigate antibacterial, antioxidant activities and total phenolic contents of five Thai medicinal plants using by Thai traditional doctors to treat opportunistic infections of AIDs patients such as Dioscorea bulbifera L. (DB), Momordica charantia L. (MC), Caricapapaya L. (female and male trees, CPF and CPM) and Phyllanthus amarus Schum & Thonn. (PA). MATERIAL AND METHOD: The ethanolic and water extracts of those herbs were tested. For antioxidant method was measured using DPPH radical scavenging assay, anti-microbial activity using disc diffusion assay and minimal inhibitory concentration (MIC) was determined by using the modified resazurin assay against four species of micro-organisms: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Candida albicans. Total phenolic content was determined by Folin-Ciocalteau colorimetric method. RESULTS: For water extract of PA showed the highest antibacterial activity against S. aureus (MIC value = 0.625 mg/ml). The ethanolic extract of MC showed the highest activity against B. subtilis (MIC = 0.625 mg/ml). Only ethanolic extract of DB inhibited growth of E. coli (MIC = 5 mg/ml) it also inhibited growth of gram positive bacteria such as S. aureus and B. subtilis with the same MIC values (2.5 mg/ml). No plant extracts showed activity against C. albicans. The ethanolic extract of CPM, PA and DB and the water extract of PA showed high antioxidant activity (EC50 of 8.48, 9.54, 11.07 and 11.37 microg/ml, respectively). The water extract of PA and the ethanolic extract of DB, CPM showed high total phenolic content of 262.54, 106.26 and 83.78 mg/g, respectively. The total phenolic content of these extracts correlated with DPPH radical scavenging activity, while only ethanolic extract of PA showed high antioxidant activity (9.54 microg/ml) but it contain low phenolic content (45.50 mg/g). CONCLUSION: Our findings support the usage of the these plants to treat opportunistic infection of Thai traditional doctors in AIDs patients. Thus, it is recommended that the isolation of pure active antibacterial and antioxidant from these plant extracts should be carried on.