Assembly of ASK1-MKK4-JNK3 Complex

Apoptosis signal-regulating kinase I (ASK1, also called MAP3K5) is a mitogen-activated kinase kinase kinase (MAP3K) that plays pivotal roles in cellular stress and immune response. Active ASK1 can directly phosphorylate downstream mitogen-activated kinase kinases (MKKs) from both stress-activated protein kinase (SAPK) pathways: c-jun N-terminal kinase (JNK). The structural of these three kinase complexes (JNK, MKK4, and ASK1) remains still unknown. For this reason, the present study has been designed to find out the assembly of ASK1-MKK4-JNK3 firstly using computational docking based on the Protein Frustratometer combined with the sequence identity calculations of Evolutionary Trace forms along with rigid docking (Cluspro) followed by experimental design. To verify the proposed computational model of ASK1-MKK4-JNK3 complex, we designed a series of maltose binding protein (MBP) fusion peptides, which contains different peptide fragments derived from MKK4: MBP-P1, MBP-P2, MBP-P3, MBP-P4 and MBP-P5. We then transformed into E-coli-BL21- DE3 to express those proteins via Isopropyl β-D-1-thiogalactopyranoside (IPTG) and then subsequently purified those proteins using Amylose-sepharose chromatography. We intend to develop direct pull-down assays to evaluate the interactions between these fusion peptides with ASK1 and JNK3, respectively to find out the binding interaction among these purified proteins

Antinociceptive and sedative activity of Vernonia patula and predictive interactions of its phenolic compounds with the cannabinoid type 1 receptor

When tested in the acetic acid-induced writhing and formalin-induced paw-licking tests, the ethanol extract of Vernonia patula (VP) aerial parts showed significant antinociceptive activity. In neuropharmacological tests, it also significantly delayed the onset of sleep, increased the duration of sleeping time, and significantly reduced the locomotor activity and exploratory behaviour of mice. Five phenolic compounds, namely gallic acid, vanillic acid, caffeic acid, quercetin and kaempferol, were detected in VP following HPLC-DAD analysis. The presence of these phenolic compounds in VP provides some support for the observed antinociceptive and sedative effects. A computational study was performed to predict the binding affinity of gallic acid, vanillic acid, caffeic acid, quercetin and kaempferol towards the cannabinoid type 1 (CB1) receptor. Caffeic and vanillic acid showed the highest probable ligand efficiency indices towards the CB1 target. Vanillic acid displayed the best blood brain barrier penetration prediction score. These findings provide some evidence for the traditional use of VP to treat pain

Ficus hispida Bark Extract Prevents Nociception, Inflammation, and CNS Stimulation in Experimental Animal Model

Background. Ficus hispida is traditionally used in the ailment of pain, inflammation, and neurological disorders. The present study set out to evaluate the in vivo antinociceptive, anti-inflammatory, and sedative activity of the ethanol extract of Ficus hispida bark (EFHB). Methods. The antinociceptive activity of EFHB was evaluated by using acetic acid induced writhing, formalin, hot plate, and tail immersion methods in Swiss albino mice. Its anti-inflammatory activity was assessed by using carrageenan and histamine induced rat paw oedema test in Wister rats. The central stimulating activity was studied by using pentobarbital induced hypnosis, hole cross, and open field tests in Swiss albino mice. Results. EFHB demonstrated antinociceptive activity both centrally and peripherally. It showed 62.24% of writhing inhibition. It significantly inhibited licking responses in early (59.29%) and late phase (71.61%). It increased the reaction time to the thermal stimulus in both hot plate and tail immersion. It inhibited the inflammation to the extent of 59.49%. A substantial increase in duration of sleep up to 60.80 min and decrease of locomotion up to 21.70 at 400 mg/kg were also observed. Conclusion. We found significant dose dependent antinociceptive, anti-inflammatory, and sedative properties of EFHB in experimental animal models

Antibacterial, cytotoxic, analgesic and diuretic activities of <i style="mso-bidi-font-style:normal">Rhizophora mucronata</i> Lam. bark

229-232<i style="mso-bidi-font-style: normal">Rhizophora mucronata Lam., locally known as ‘Garjon or Jhanna’ in Bangladesh is a mangrove plant of Rhizophoraceae family. Different parts of this plant have got applications in folk medicine from the ancient time. In the present study, attempts have been made for a detailed phytochemical study of the bark of the plant for antibacterial, cytoxic, analgesic and diuretic activites. Disc diffusion method was used for the screening of antibacterial activity and cytotoxicity study was performed by brine shrimp lethality bioassay. Acetic acid induced writhing method was employed to assess the analgesic potentiality of the extract. Diuretic property of the bark extract was studied by Lipschitz method. The ethanolic extract of the barks of R. mucronata Lam. showed moderate antibacterial activity against both Gram-positive and Gram-negative strains. Marked inhibitory effects were found with Escherichia coli and Staphylococus epidermidis at a concentration of 500 µg/disc. The extract showed very high level of general toxicity in the brine shrimp lethality bioassay having an LC50 value of 0.5 µg/mL. The extract of <i style="mso-bidi-font-style: normal">R. mucronata Lam. bark at concentrations of 250 mg/kg and 500 mg/kg exhibited significant (P<0.01) inhibition of writhing reflex by 36.96% and 50%, respectively while the standard diclofenac inhibition was found to be 65.21% at a dose of 50 mg/kg body weight. The extract also showed very high response to diuresis in a dose-dependent manner

Polyphenolic Compounds Inhibit Osteoclast Differentiation While Reducing Autophagy through Limiting ROS and the Mitochondrial Membrane Potential

Polyphenolic compounds are a diverse group of natural compounds that interact with various cellular proteins responsible for cell survival, differentiation, and apoptosis. However, it is yet to be established how these compounds interact in myeloid cells during their differentiation and the molecular and intracellular mechanisms involved. Osteoclasts are multinucleated cells that originate from myeloid cells. They resorb cartilage and bone, maintain bone homeostasis, and can cause pathogenesis. Autophagy is a cellular mechanism that is responsible for the degradation of damaged proteins and organelles within cells and helps maintain intracellular homeostasis. Imbalances in autophagy cause various pathological disorders. The current study investigated the role of several polyphenolic compounds, including tannic acid (TA), gallic acid (GA), and ellagic acid (EA) in the regulation of osteoclast differentiation of myeloid cells. We demonstrated that polyphenolic compounds inhibit osteoclast differentiation in a dose-dependent manner. Quantitative real-time PCR, immunocytochemistry, and western blotting revealed that osteoclast markers, such as NFATc1, Cathepsin K, and TRAP were inhibited after the addition of polyphenolic compounds during osteoclast differentiation. In our investigation into the molecular mechanisms, we found that the addition of polyphenolic compounds reduced the number of autophagic vesicles and the levels of LC3B, BECN1, ATG5, and ATG7 molecules through the inactivation of Akt, thus inhibiting the autophagy process. In addition, we found that by decreasing intracellular calcium and decreasing ROS levels, along with decreasing mitochondrial membrane potential, polyphenolic compounds inhibit osteoclast differentiation. Together, this study provides evidence that polyphenolic compounds inhibit osteoclast differentiation by reducing ROS production, autophagy, intracellular Ca2+ level, and mitochondrial membrane potentials