Phenolic Extraction of Moringa Oleifera Leaves Induces Caspase-Dependent and Caspase-Independent Apoptosis through the Generation of Reactive Oxygen Species and the Activation of Intrinsic Mitochondrial Pathway in Human Melanoma Cells.

Moringa oleifera Lam. has long been used to treat many diseases, including diabetes, aging, inflammatory, and cancer. Many studies have revealed that the crude extract of Moringa oleifera Lam. leaves possesses anticancer property. Therefore, in this study, the extract of Moringa oleifera leaves was fractionated using different solvents to figure out the most effective fraction for anti-proliferative effect on melanoma cells. Methanol extract (MO-ME), hexane fraction (MO-HE), chloroform fraction (MO-CH), ethyl acetate fraction (MO-EA), and water-soluble fraction (MO-WA) of Moringa oleifera leaves were prepared. Total phenolic and flavonoid contents were determined. The anti-proliferative activity on melanoma cells and normal cells was investigated using WST-1 assay. The apoptotic activity was assessed by testing DNA condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The expression of apoptosis-related genes, the mitochondrial depolarization, and reactive oxygen species (ROS) were then examined to clarify the underlying molecular mechanisms. In this regard, MO-ME, MO-EA, and MO-CH inhibited the proliferation of both A375 human melanoma cells and A2058 human melanoma cells, but had little effect on WS1 normal human skin fibroblasts and primary normal human dermal fibroblasts (NHDF). Among fractions, the phenolic-rich MO-EA markedly inhibited the growth of A375 cells in a dose- and time-dependent manner. The anti-proliferation was supposed to be mediated via apoptosis, which was demonstrated by the significant increase of condensed chromatin, DNA fragmentation, and PS externalization. The apoptosis was stimulated by enhanced ROS production and reduction of mitochondrial membrane potential. MO-EA activated Bax while reducing Bcl-2 expression, leading to an increase in Bax/Bcl-2 ratio. The mechanisms of cell death involved in activation of Caspase-3/7 and Caspase-9 (Caspase-dependent pathway), activation, and translocation of apoptosis-inducing factor (AIF) into the nucleus (Caspase-independent pathway). Our study indicated that the phenolic-rich fraction exerted significant anticancer effects on melanoma cells in vitro which involved in Caspase-dependent and Caspase-independent apoptosis pathways mediated by mitochondrial ROS. These results provided a fundament for the using of phenolic-rich fraction of Moringa oleifera leaves to treat skin cancer effectively.

Smad linker region phosphorylation is a signalling pathway in its own right and not only a modulator of canonical TGF-ß signalling.

Transforming growth factor (TGF)-ß signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-ß signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-ß receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-ß can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-ß signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-ß signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.

Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase.

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.

Mitochondria-mediated Caspase-dependent and Caspase-independent apoptosis induced by aqueous extract from Moringa oleifera leaves in human melanoma cells.

Malignant melanoma is a very aggressive and serious type of cutaneous cancer. Previous studies indicated the anti-cancer activity of aqueous extract of Moringa oleifera Lam. leaves (MOE) against a variety of cell lines. However, there has not been much research about the effect of MOE on melanoma. Therefore, this study was about to investigate the anti-proliferation mediated by apoptosis of MOE on human melanoma cell lines. Furthermore, the related molecular mechanisms of the apoptosis were also examined. An aqueous extract of Moringa oleifera leaves was prepared and the anti-proliferative activity on melanoma cells and normal cells was tested using WST-1 assay. The apoptotic hallmarks including DNA condensation and phosphatidylserine (PS) externalization were assessed. The expression of apoptosis-related genes and the depolarization of mitochondrial membrane potential were then examined to clarify the underlying molecular mechanisms. MOE inhibited cell growth of A375 cells and A2058 cells in a dose-dependent manner but had little effect on human normal fibroblasts. The cell growth inhibition was induced by apoptosis which was expressed via chromatin condensation and PS externalization. MOE decreased mitochondrial membrane potential. Additionally, MOE increased Bax/Bcl-2 ratio, activated Caspase-3/7, Caspase-9, PARP and AIF translocation, leading to apoptotic cell death. Our study indicated that MOE exerted significant anti-cancer effects on melanoma cells in vitro which involved mitochondria-mediated Caspase-dependent and Caspase-independent apoptosis pathways. These results provided a scientific approach for using Moringa oleifera leaves as an alternative therapy to treat skin cancer.

Soluble expression and partial purification of recombinant human erythropoietin from E. coli.

Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.

Chemical composition and the anti-inflammatory effect of volatile compounds from Anaxagorea luzonensis A. Gray.

Anaxagorea luzonensis A. Gray, a member of the Annonaceae family, has been used to treat a variety of illnesses for a long time. For the first time, A. luzonensis volatile compounds (ALVCs) were extracted from the leaves, and the components were identified using gas chromatography-mass spectrometry (GC-MS). Further, the main compositions of ALVCs were also assessed for their ability to bind with anti-inflammatory proteins using a docking model. In addition, in vitro tests e.g. inhibition of protein degradation and the inhibition of nitric oxide release using RAW264.7 macrophage cells were utilized for evaluating the anti-inflammatory activity. The results showed that the principal compounds of ALVCs were bulnesol (34.1 %), cubitene (17.8 %), ß-eudesmol (10.4 %), epi-longipinanol (5.9 %), and (Z)-nerolidyl acetate (5.5 %). Three compounds viz. bulnesol, cubitene, and ß-eudesmol bound firmly to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), as shown by the in silico analysis, similar to the positive control diclofenac. ALVCs effectively inhibited protein degradation with the IC50 of 31 ± 2.3 µg/mL and inhibited nitric oxide production with the IC50 of 43.30 ± 3.37 µg/mL. These findings showed that ALVCs might have a promising anti-inflammatory effect by blocking several inflammatory proteins.

Chemical Composition, Anti-bacterial Activity and Molecular Docking Studies of Essential Oil Isolated from Sa Sam Nam (Launaea sarmentosa).

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Loss-of-Function Mutation of ACTIN-RELATED PROTEIN 6 (ARP6) Impairs Root Growth in Response to Salinity Stress.

H2A.Z-containing nucleosomes have been found to function in various developmental programs in Arabidopsis (e.g., floral transition, warm ambient temperature, and drought stress responses). The SWI2/SNF2-Related 1 Chromatin Remodeling (SWR1) complex is known to control the deposition of H2A.Z, and it has been unraveled that ACTIN-RELATED PROTEIN 6 (ARP6) is one component of this SWR1 complex. Previous studies showed that the arp6 mutant exhibited some distinguished phenotypes such as early flowering, leaf serration, elongated hypocotyl, and reduced seed germination rate in response to osmotic stress. In this study, we aimed to investigate the changes of arp6 mutant when the plants were grown in salt stress condition. The phenotypic observation showed that the arp6 mutant was more sensitive to salt stress than the wild type. Upon salt stress condition, this mutant exhibited attenuated root phenotypes such as shorter primary root length and fewer lateral root numbers. The transcript levels of stress-responsive genes, ABA INSENSITIVE 1 (ABI1) and ABI2, were found to be impaired in the arp6 mutant in comparison with wild-type plants in response to salt stress. In addition, a meta-analysis of published data indicated a number of genes involved in auxin response were induced in arp6 mutant grown in non-stress condition. These imply that the loss of H2A.Z balance (in arp6 mutant) may lead to change stress and auxin responses resulting in alternative root morphogenesis upon both normal and salinity stress conditions.

The anti-inflammatory activity of the compounds isolated from Dichroa febrifuga leaves.

Dichroa febrifuga Lour. is a traditional medicinal herb that has been applied in the treatment of malaria and some other infectious diseases. Studies recently have focused on the anti-inflammation of the extracts of Dichroa febrifuga Lour. although there have not many reports about which compounds play the essential role. Therefore, in this study, we isolated hydrangenoside C (1), isoarborinol (2), and methyl 1,3,4,6-tetra-O-acetyl-fructofuranoside (3) from the leaves of Dichroa febrifuga. Subsequently, the anti-inflammatory property of 1-3 was assessed using an in vivo assay of edema mouse model which was induced by carrageenan. Out of the three, 2 inhibited the edema effectively and dose-dependently, similarly to diclofenac while there was no obvious activity observed in 1 and 3. The in silico results demonstrated that 2 enables binding to 5-LOX and PLA2 via generating h-bonds. This is the first study to mention the anti-inflammation of 2 in Dichroa febrifuga Lour., and would be a contribution to further studies to elucidate the promising bioactivities of this compound.

Preparation, Characterization, and Anti-Adhesive Activity of Sulfate Polysaccharide from Caulerpa lentillifera against Helicobacter pylori.

In the gastric mucosa, chronic inflammation due to Helicobacter pylori infection promotes gastrocarcinogenesis. Polysaccharides of Caulerpa lentillifera are well-characterized by broad antimicrobial activity and anti-inflammatory potentials. The present study was undertaken to investigate whether the low molecular sulfate polysaccharides of C. lentillifera (CLCP) exhibit any anti-adhesive activity against H. pylori. After a hot water extraction and purification process, two purified polysaccharide fractions (CLCP-1 and CLCP2) were studied based on structural characterization and bioactivity determination. The results implied that except for the molar ratio, CLCP-1 and CLCP-2 contain high sulfate, mannose, galactose, xylose, glucose levels, and low protein levels. The molecular weight and Fourier transform infrared spectroscopy (FT-IR) assays confirmed that CLCP-1 and CLCP-2 are sulfate polysaccharides with an average molecular weight (Mw) of 963.15 and 648.42 kDa, respectively. In addition, CLCP-1 and CLCP-2 exhibited stronger antibacterial activity against H. pylori. CLCP-1 and CLCP-2 could significantly promote macrophage proliferation and decrease the production of nitric oxide (NO) through downregulated expression of inducible nitric oxide synthase (iNOS). Meanwhile, CLCP-1 and CLCP-2 in this study showed efficiently protected gastric adenocarcinoma (AGS) cells against H. pylori with the inhibition of the IL-8/NF-κB axis. These findings suggested the effect of Caulerpa lentillifera polysaccharides on H. pylori adhesion, a potential supply of nutrients for eradication therapy through the reduction of cell count and inflammation.

Antinociceptive and Anti-Inflammatory Effects of Recombinant Crotamine in Mouse Models of Pain.

Crotamine, a toxin found in the venom of the South American rattlesnake Crotalus durissus terrificus, has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive and anti-inflammatory effects using the hot-plate test, acetic-acid-induced writhing method, and formalin test in mice. Recombinant crotamine was administered intraperitoneally (0.04-1.2 mg kg-1) or intraplantarly (0.9-7.5 µg 10 µL-1) before the tests. The paw volume was measured with a plethysmometer. To evaluate the antagonistic and anti-inflammatory effects of naloxone, subcutaneous naloxone (4 mg kg-1) or intraplantar naloxone (5 µg 10 µL-1) was administered before recombinant crotamine. For tumor necrosis factor (TNF)-α assays, blood was drawn 3 h after formalin injection and measured using enzyme-linked immunosorbent assay. Intraperitoneal and intraplantar recombinant crotamine had antinociceptive and anti-inflammatory effects, neither of which were affected by pre-treatment with naloxone. The mean serum TNF-α levels were significantly lower in the intraperitoneal recombinant crotamine (0.4 and 1.2 mg kg-1) or intraplantar (2.5 and 7.5 µg 10 µL-1) recombinant crotamine groups than in the saline group and were not affected by naloxone pre-treatment. In conclusion, recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear to be related to the opioid receptor. The antinociceptive and anti-inflammatory effects of intraperitoneal or intraplantar recombinant crotamine are related to TNF-α.

Antioxidant and Antimicrobial Activities of the Extracts from Different Garcinia Species.

BACKGROUND: Garcinia is a large genus which has promising bioactivities. However, the properties of many Garcinia species have not been investigated thoroughly. AIM: To determine the antioxidant and antimicrobial capabilities of the extracts from different Garcinia species. Methodology. Six Garcinia species, including Garcinia fusca, Garcinia hopii, Garcinia planchonii, Garcinia nigrolineata, Garcinia gaudichaudii, and Garcinia tinctoria were extracted using n-hexane, ethyl acetate, and methanol, producing n-hexane extract (HE), ethyl acetate extract (EAE), and methanol extract (ME). After that, the total polyphenol content was evaluated using Folin-Ciocalteu assay. DPPH, hydroxyl radical scavenging, and total antioxidant capacity assays were performed to test the antioxidant activity. Subsequently, the antimicrobial activities against Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacterial strains were assessed using Kirby Bauer and the broth microdilution methods. RESULTS: Many Garcinia extracts contained high total polyphenol content consisting of ME of G. hopii ad G. tinctoria, and EAE of G. planchonii and G. tinctoria. The EAE of G. tinctoria showed effective antioxidant capacity (IC50 = 1.5 µg/mL). Additionally, the EAE of G. gaudichaudii was effective against Gram-positive bacteria with minimal inhibition concentration (MIC) of 15.625-25 µg/mL whereas ME of G. planchonii was effective against both Gram-positive bacteria (MIC = 160 µg/mL) and Gram-negative bacteria (MIC = 75 µg/mL). CONCLUSION: Several extracts of Garcinia species demonstrated valuable antioxidant and antimicrobial properties.

Anti-Inflammatory and Antimicrobial Activities of Compounds Isolated from Distichochlamys benenica.

Distichochlamys benenica is a native black ginger that grows in Vietnam. In point of fact, there is limitation of available information in the literature making mention of the chemical constituents and bioactive properties of this plant. This study is aimed at isolating trans-o-coumaric acid (1), trans-cinnamic acid (2), and borneol (3) from the rhizomes of D. benenica Q.B.Nguyen & Skornick and evaluate the anti-inflammatory and antimicrobial activities of 1-3 using the carrageenan paw edema model and the dilution broth method, respectively. This revealed that 1 was as effective as diclofenac in reducing the intensity of the edema development. The in silico research showed that the activity of 1 might be derived from inhibiting COX-2 by generating h-bonds at the positions of Arg 120, Tyr 355, and Arg 513 residues. The antimicrobial activities against Gram-positive strains (Staphylococcus aureus and Bacillus subtilis) were comparable, with the minimum inhibitory concentrations ranging from 1.52 to 3.37 mM. This is the first study of the bioactivity of compounds isolated from D. benenica Q.B.Nguyen & Skornick. Our results suggest that 1 may be a nature-derived compound which demonstrates the anti-inflammatory properties and inhibit the proliferation of several Gram-positive bacteria.

Vernolide-A and Vernodaline: Sesquiterpene Lactones with Cytotoxicity against Cancer.

Vernolide-A and vernodaline are sesquiterpene lactones isolated from genera of Vernonia. Vernolide-A and vernodaline have shown promising therapeutic properties, including antibacterial, antihelminth, and antioxidant activities. Recently, the anticancer properties of these sesquiterpene lactones have been investigated with the elucidation of effects on cell proliferation, metastasis, angiogenesis, and apoptosis. The antiproliferation and antimetastatic activities arise from targeting extracellular signal-regulated kinase 1 (ERK-1), extracellular signal-regulated kinase 2 (ERK-2), nuclear factor-κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase 2 (MMP-2), and matrix metalloproteinase 9 (MMP9). The induction of apoptosis is due to the enhancement of caspase 9, caspase 3, while inhibition of Bcl-2 and Bcl-xL results in the release of cytochrome c into the cytosol. The activity of vernolide-A and vernodaline is hypothesized to be due to thiol reactivity through the α-methylene-γ-lactone group of sesquiterpene lactones. This review will give a brief summary of the anticancer activity of vernolide-A and vernodaline and provide information on the underlying molecular mechanisms.

Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains.

Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b'a' domain of PDI (PDIb'a'), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/µg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC50 and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

Correction: Prokaryotic soluble overexpression and purification of human VEGF165 by fusion to a maltose binding protein tag.

[This corrects the article DOI: 10.1371/journal.pone.0156296.].

Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF.

Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with the Fc domain of IgG1 at the C terminus (GCSF-Fc) and with the maltose binding protein (MBP) tag at the N-terminus and expressed it as a soluble protein in the cytoplasm of E. coli. We also conjugated PEG aldehyde to GCSF to make PEG-GCSF. The bioactivities of GCSF-Fc and PEG-GCSF were similar to native GCSF using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50 dose-response curves for GCSF, GCSF-Fc and PEG-GCSF were 37 ± 12 pM, 75 ± 13.5 pM and 46 ± 5.5 pM, respectively. When the proteins were injected into neutropenic rats, the group injected with PEG-GCSF showed the highest and fastest recovery of neutrophils, followed by GCSF-Fc and GCSF. ELISA assay revealed the PEG-GCSF had the longest plasma circulation (>72 h), followed by GCSF-Fc (>48 h) and GCSF (~24 h), which is consistent with the in vivo activities of the proteins. In summary, the GCSF-Fc purified from E. coli was not as efficient as PEG-GCSF in treating neutropenic rats.

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein.

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a ß-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.

Crotamine stimulates phagocytic activity by inducing nitric oxide and TNF-α via p38 and NFκ-B signaling in RAW 264.7 macrophages.

Crotamine is a peptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus and has antiproliferative, antimicrobial, and antifungal activities. Herein, we show that crotamine dose-dependently induced macrophage phagocytic and cytostatic activity by the induction of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). Moreover, the crotamineinduced expression of iNOS and TNF-α is mediated through the phosphorylation of p38 and the NF-κB signaling cascade in macrophages. Notably, pretreatment with SB203580 (a p38-specific inhibitor) or BAY 11-7082 (an NF-κB inhibitor) inhibited crotamine-induced NO production and macrophage phagocytic and cytotoxic activity. Our results show for the first time that crotamine stimulates macrophage phagocytic and cytostatic activity by induction of NO and TNF-α via the p38 and NF-κB signaling pathways and suggest that crotamine may be a useful therapeutic agent for the treatment of inflammatory disease. [BMB Reports 2016; 49(3): 185-190].