Regulatory role of oligodendrocyte gap junctions in inflammatory demyelination.

Gap junctions (GJs) coupling oligodendrocytes to astrocytes and to other oligodendrocytes are formed mainly by connexin47 (Cx47) and a smaller portion by connexin32 (Cx32). Mutations in both connexins cause inherited demyelinating disorders, but their expression is also disrupted in multiple sclerosis (MS). To clarify whether the loss of either Cx47 or Cx32 could modify the outcome of inflammation and myelin loss, we induced experimental autoimmune encephalomyelitis (EAE) in fully backcrossed Cx32 knockout (KO) and Cx47KO mice and compared their outcome with wild type (WT, C57BI/6 N) mice. Cx47KO EAE mice developed the most severe phenotype assessed by clinical scores and behavioral testing, followed by Cx32KO and WT mice. Cx47KO more than Cx32KO EAE mice developed more microglial activation, myelin, and axonal loss than did WT mice. Oligodendrocyte apoptosis and precursor proliferation was also higher in Cx47KO than in Cx32KO or WT EAE mice. Similarly, blood-spinal cord barrier (BSCB) disruption and inflammatory infiltrates of macrophages, T- and B-cells were more severe in Cx47KO than either Cx32KO or WT EAE groups. Finally, expression profiling revealed that several proinflammatory cytokines were higher at the peak of inflammation in the Cx47KO mice and persisted at later stages of EAE in contrast to reduction of their levels in WT EAE mice. Thus, loss of oligodendrocyte GJs aggravates BSCB disruption and inflammatory myelin loss, likely due to dysregulation of proinflammatory cytokines. This mechanism may play an important role in MS brain with reduced connexin expression, as well as in patients with inherited mutations in oligodendrocyte connexins and secondary inflammation.

Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review.

The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guarantee an unlimited supply of recombinant proteins. The demand of recombinant proteins has increased as more applications in several fields become a commercial reality. Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, producing soluble proteins in E. coli is still a major bottleneck for structural biology projects. One of the most challenging steps in any structural biology project is predicting which protein or protein fragment will express solubly and purify for crystallographic studies. The production of soluble and active proteins is influenced by several factors including expression host, fusion tag, induction temperature and time. Statistical designed experiments are gaining success in the production of recombinant protein because they provide information on variable interactions that escape the "one-factor-at-a-time" method. Here, we review the most important factors affecting the production of recombinant proteins in a soluble form. Moreover, we provide information about how the statistical design experiments can increase protein yield and purity as well as find conditions for crystal growth.

Rationally designed less toxic SPD-304 analogs and preliminary evaluation of their TNF inhibitory effects.

SPD-304 was discovered as a promising tumor necrosis factor alpha (TNF) antagonist that promotes dissociation of TNF trimers and therefore blocks the interaction of TNF and its receptor. However, SPD-304 contains a potentially toxic 3-alkylindole moiety, which can be bioactivated to a reactive electrophilic intermediate. A series of SPD-304 analogs was synthesized with the aim to diminish its toxicophore groups while maintaining the binding affinity for TNF. Incorporation of electron-withdrawing substituents at the indole moiety, in conjunction with elimination of the 6'-methyl group of the 4-chromone moiety, led to a significantly less toxic and equally potent TNF inhibitor.

A statistical approach for optimization of RANKL overexpression in Escherichia coli: purification and characterization of the protein.

Receptor activator of nuclear factor-κB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3mM, a post-induction temperature of 25°C and a post-induction time of 6.5h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-α, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1 ± 0.5 µM.

Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies.

Tumor necrosis factor-α (TNF-α) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-α is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-α inhibitors. In this work, induction conditions for the production of human TNF-α fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD(600nm)), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-α. An 11% increase of TNF-α production was achieved after determination of the optimum induction conditions: OD(600nm) prior induction 0.55, a post induction temperature of 25°C, an IPTG concentration of 1mM and a post-induction time of 4h. We have also studied TNF-α oligomerization, the major property of this protein, and a K(d) value of 0.26nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable K(d) value for protein trimerization probably due to the complexibility of our model.

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility.

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.

An extracellular polyhydroxybutyrate depolymerase in Thermus thermophilus HB8.

The thermophilic bacterium Thermus thermophilus HB8 has been characterized as a polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and forms clear zones on agar plates containing PHB as sole carbon source. T. thermophilus extracellular PHB depolymerase was purified to homogeneity using an affinity chromatography protocol. The purified enzyme was estimated to have an apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was identified as the TTHA0199 gene product of T. thermophilus HB8. The amino acid sequence of the TTHA0199 gene product shared significant homologies to other carboxylesterases. A catalytic triad was identified consisting of S(183), E(310), and H(405). A pentapeptide sequence (GX(1)SX(2)G) exists within the molecule, characteristic for PHB depolymerases (lipase box) and for other serine hydrolases. Purified extracellular PHB depolymerase was stable at high temperatures with an optimum activity at pH 8.0. The apparent Km value of the purified enzyme for PHB was 53 microg/ml. As the main product of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that the enzyme acts principally as an exo-type hydrolase.

Quantification of the effects of ionic strength, viscosity, and hydrophobicity on protein-ligand binding affinity.

In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (K d) is essential. Estimation of the binding affinity in this way is routinely performed in "favorable" conditions for macromolecules. Crucial data for ligand-protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the K d of three previously characterized protein-ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity.

Solvent Selection for Insoluble Ligands, a Challenge for Biological Assay Development: A TNF-α/SPD304 Study.

Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-α inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-α/SPD304 system, and a K d of 5.36 ± 0.21 µM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins.

Simultaneous polyhydroxyalkanoates and rhamnolipids production by Thermus thermophilus HB8.

The ability of Thermus thermophilus HB8 to produce simultaneously two environmentally-friendly biodegradable products, polyhydroxyalkanoates (PHAs) and rhamnolipids (RLs), using either sodium gluconate or glucose as sole carbon source, was demonstrated. The utilization of sodium gluconate resulted in higher levels of PHAs and RLs production than when glucose was used as sole carbon source. The initial phosphate concentration (as PO43-) influences both PHAs and RLs productions that were increased during cultivation time. PHAs accumulation was enhanced (> 300 mg/L) after 72 h of cultivation in an initial [PO43-] of 25 mM, while RLs production (> 200 mg/L) was started after 35 h and continued until 72 h of cultivation, in a phosphate-limited medium containing initially 5 mM of [PO43-]. In addition, the combine effect of initial [PO43-] and cultivation time on biomass, PHAs and RLs production was evaluated from 2D contour plots. The results revealed that low initial phosphate concentrations (up to 5 mM) and long incubation time (72 h) promoted RLs biosynthesis while higher initial phosphate concentrations (up to 25 mM) where favorable for biomass and PHAs production. The molecular composition of the produced bio-products was identified. The accumulated PHAs were co-polymers which mainly consisted of 3-hydroxydecanoate (3HD) as resulted by gas chromatography (GC) analysis. The secreted RLs were extracted and their total mixture contained both mono- and di- RLs identified by thin-layer chromatography (TLC). Moreover, the molecular composition of the produced RLs characterized in details by LC-MS analysis showed a plethora of diversity including mono-, and di-RLs, di-rhamno-monolipidic congeners differing in the length of the lipidic chain, which additionally were found to be saturated or unsaturated in some cases.