A designer hyper interleukin 11 (H11) is a biologically active cytokine

<p>Abstract</p> <p>Background</p> <p>Interleukin 11 (IL-11) is a pleiotropic cytokine with anti-apoptotic, anti-inflammatory and hematopoietic potential. The IL-11 activity is determined by the expression of the IL-11R receptor alpha (IL-11Rα) and the signal transducing subunit β (gp130) on the cell membrane. A recombinant soluble form of the IL-11Rα (sIL-11Rα) in combination with IL-11 acts as an agonist on cells expressing the gp130 molecule. We constructed a designer cytokine Hyper IL-11 (H11), which is exclusively composed of naturally existing components. It contains the full length sIL-11Rα connected with the mature IL-11 protein using their natural sequences only. Such a construct has two major advantages: (i) its components are as close as possible to the natural forms of both proteins and (ii) it lacks an artificial linker what should avoid induction of antibody production.</p> <p>Results</p> <p>The H11 construct was generated, the protein was produced in a baculovirus expression system and was then purified by using ion exchange chromatography. The H11 protein displayed activity in three independent bioassays, (i) it induced acute phase proteins production in HepG2 cells expressing IL-11, IL-11Rα and gp130, (ii) it stimulated the proliferation of B9 cells (cells expressing IL-11Rα and gp130) and (iii) proliferation of Baf/3-gp130 cells (cells not expressing IL-11 and IL-11Rα but gp130). Moreover, the preliminary data indicated that H11 was functionally distinct from Hyper-IL-6, a molecule which utilizes the same homodimer of signal transducing receptor (gp130).</p> <p>Conclusions</p> <p>The biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.</p

Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase

A Ligand Peptide Motif Selected from a Cancer Patient Is a Receptor-Interacting Site within Human Interleukin-11

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11Rα is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11Rα has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs

Simultaneous Detection of Whole Blood Chemiluminescence in Microtitre Plates

Peer Reviewe

Picosecond fluorescence microscopy for measuring chlorophyll and porphyrin components in conifers and cultured cells.

A synchroscan streak camera was adapted to a microscopic system for fluorescence lifetime measurements. By using this device together with a synchronously pumped dye laser system, a time resolution of 40 ps was attained. Recent applications of this technique include the fluorometric detection of chlorophyll from microscopic parts of spruce needles to measure defects within the photosynthetic system after exposition to environmental pollutants or their photooxidants and fungal infection. It was found that after a combination of continuously high ozone concentration and infection by Rhizosphaera Kalkhoffii, the relative fluorescence intensity of a chlorophyll component with a decay time of 550 ps decreased significantly, whereas a component decaying with 110 ps remained almost constant. This indicates a selective inhibition within the photosystem II. In addition, fluoresence lifetimes of hematoporphyrin derivative - a tumor-selective photosensitizer used for diagnosis and photodynamic therapy of cancer - were measured in the picosecond time range. In comparison with previous nanosecond measurements, a new fluorescent component withh a lifetime of 90 ps was detected from Hpd solutions. This component may be attributed to a dimeric or aggregated species which is monomerized when taken up by malignant or non-malignant cells