E. coli expression and purification of human and cynomolgus IL-15

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)₆-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His)₆-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3⁻ CD8⁺ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.     

Synthesis, characterization, spectroscopy, electronic and redox properties of a new nickel dithiolene system

A new dithiolene ligand with 3,5-dibromo substituted phenyl groups was designed and synthesized. The protected form of the ligand was reacted with a nickel salt providing neutral Ni(S₂C₂(3,5-C₆H₃Br₂)₂)₂ and anionic [Ni(S₂C₂(3,5-C₆H₃Br₂)₂)₂]⁻ isolated as a Bu₄N⁺ salt. Both were characterized by UV-Vis and IR spectroscopy and compared with the similar known molecular systems. They exhibit intense low energy transitions that are characteristic of such systems. The electrochemical behavior of these molecules was investigated by cyclic voltammetry.     

Syntheses, characterizations and theoretical calculations of rhodium(III) 1,2-naphthoquinone-1-oxime complexes

Rhodium(III) complexes of 1,2-naphthoquinone-1-oxime (1-nqo) [Rh(1-nqo)L₂Cl₂] 1-3 [1, L = 4-methylpyridine (mpy); 2, L = 4-phenylpyridine (ppy); 3, L = 4-acetylpyridine (apy)] were prepared. The structure of complex 1 is analyzed by single crystal X-ray crystallography. All of the complexes were characterized by mass spectrometry, ¹H-¹H COSY NMR and FT-IR. UV-Vis absorption spectroscopy and cyclic voltammetry were employed to investigate the electronic transition behaviors of the complexes. The complexes displayed irreversible metal-localized two-electron reductions from Rh^III to Rh^I on the cyclic voltammogram. While the low-energy absorptions at λ_max of 488-490 nm on the UV-Vis spectra of the complexes were related to metal to 1-nqo ligand charge transfer [MLCT, dπ(Rh) → π∗(1-nqo)] and chloride to 1-nqo ligand charge transfer [LLCT, pπ(Cl) → π∗(1-nqo)] based on the theoretical calculations using time-dependent density functional theory (TD-DFT).     

Photochemical and photophysical properties of ruthenium(II) bis-bipyridine bis-nitrile complexes: Photolability

The electrochemical and photophysical properties of two bis-nitrilo ruthenium(II) complexes formulated as [Ru(bpy)₂(L)₂](PF₆)₂, where bpy is 2,2′-bipyridine and L is AN = CH₃CN and sn = NC-CH₂CH₂-CN, have been investigated. Electrochemical data are typical of Ru-bpy complexes with two reversible reduction peaks located near −1.3 and −1.6 V assigned to each bipyridine ligand and one Ru^II/Ru^III oxidation wave centered at approximately +1.5 V. The sn derivative is both IR and Raman active with its coordinated CN stretch appearing at 2277 cm⁻¹ and 2273 cm⁻¹, respectively. The UV/Vis absorption spectrum of the sn derivative is dominated by an intense (ε_max ∼ 58700 M⁻¹ cm⁻¹) absorption band at 287 nm assigned as a LC (π → π∗) transition. The peak observed at 418 nm (ε ∼ 10 400 M⁻¹ cm⁻¹) is an MLCT band while the one at 244 nm (ε ∼ 23 600 M⁻¹ cm⁻¹) is of LMLCT character. The AN derivative behaves similarly. Both complexes show low-temperature emission at around 537 nm with a lifetime near 10.0 μs. ¹H and ¹³C assignments are consistent with the formulation of the complexes. The complexes undergo photosubstitution of solvent with quantum efficiencies near one. Calculated and experimental results support replacement of the nitrile ligands by solvent. Based on DFT calculations, the electron density of the HOMO lies on the metal center, the bipyridine ligands and the nitrile ligands and electron density of the LUMO resides primarily on the bipyridine ligands. The electronic spectra obtained from TDDFT calculations closely match the experimental ones.     

The molecular and electronic structures of monomeric cobalt complexes containing redox noninnocent o-aminobenzenethiolate ligands

Dark blue [PPh₄][Co^III(²L)] (2), where (²L)²⁻ represents the closed-shell dianion of 4,6-di-tert-butyl-2-[(pentafluorophenyl)amino]benzenethiol, has been synthesized from the reaction of H₂(²L) and CoCl₂ (2:1) in acetonitrile with excess NEt₃, brief exposure of the solution to air, and addition of [PPh₄]Br. The oxidation of 2 with one equivalent of iodine produces the neutral species [Co^III(²L)₂I]⁰ (3), where (²L)¹⁻ represents the one-electron oxidized π radical anion of (²L)²⁻. Crystalline [Co^III(⁴L)] (4), where (⁴L)³⁻ is the π radical monoanion of bis-2,2′-(1,2-diphenylethylenediimine)-benzenethiolate, was precipitated from a toluene reflux of [Co^II(³L)₂], where (³L)²⁻ is the closed-shell monoanion of 2-(phenylmethylamino)benzenethiol. The reduction of 4 with CoCp₂ under anaerobic conditions yielded dark violet crystals of [CoCp₂][Co^III(⁴L)] (5). The reaction of Zn(CH₃CO₂)₂ with 2-phenylbenzothiazoline in methanol resulted in the formation of [Zn^II(³L)₂]⁰ (6). The two monoanions 2, and 5, along with [N(n-Bu)₄][Co(abt)₂] (1) (abt²⁻ = o-aminobenzenethiolate), and neutral 4 have all been shown by X-ray crystallography to be square planar. A tetrahedral geometry was adopted by 6. From temperature dependent (3-300 K) magnetic susceptibility measurements, it was established the monoanions have a triplet ground state characterized by a large zero field splitting. EPR measurements of 4, and electrochemically oxidized 1 and 2 reveal distinctly different spin Hamiltonian parameters that are interpreted with the aid of density function theoretical (DFT) calculations. It is shown that oxidation states describing a d⁶ Co(III) or d⁷ Co(II) cannot be unambiguously assigned for these neutral and monoanionic species.     

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

南美斑潜蝇在温室蔬菜上种群动态的初步观察

系统观察结果表明,南美斑潜蝇Liriomyza huidobrensis(Blanchard)在平凉市日光温室蔬菜全生产期内的种群消长明显呈“两头高中间低”的态势,成虫发生量以春夏生产期最大,可达2.1头/叶;深冬生产期最小,仅为0.1~0.2头/叶;秋冬生产期较大,保持在1.0~1.2头/叶之间。温室内温、湿度条件的变化是引起该虫种群数量波动的主要原因。根据有效积温计算结果,该虫在平凉市日光温室蔬菜全生产期发生9.1代,世代平均发育速率0.030,不同生产期的发生代数和发育速率差异较大。生产上宜采取“前控后治中间松”的防治策略。     

Model-based analysis and design of a microchannel reactor for tissue engineering

Recently developed perfusion micro-bioreactors offer the promise of more physiologic in vitro systems for tissue engineering. Successful application of such bioreactors will require a method to characterize the bioreactor environment required to elicit desired cell function. We present a mathematical model to describe nutrient/growth factor transport and cell growth inside a microchannel bioreactor. Using the model, we first show that the nature of spatial gradients in nutrient concentration can be controlled by both design and operating conditions and are a strong function of cell uptake rates. Next, we extend our model to investigate the spatial distributions of cell-secreted soluble autocrine/paracrine growth factors in the bioreactor. We show that the convective transport associated with the continuous cell culture and possible media recirculation can significantly alter the concentration distribution of the soluble signaling molecules as compared to static culture experiments and hence needs special attention when adapting static culture protocols for the bioreactor. Further, using an unsteady state model, we find that spatial gradients in nutrient/growth factor concentrations can bring about spatial variations in the cell density distribution inside the bioreactor, which can result in lowered working volume of the bioreactor. Finally, we show that the nutrient and spatial limitations can dramatically affect the composition of a co-cultured cell population. Our results are significant for the development, design, and optimization of novel micro-channel systems for tissue engineering.     

Effect of substrate concentration on dual-species biofilm population densities of Klebsiella oxytoca and Burkholderia cepacia in porous media

The long-term operation of bioremediation technologies relies on the success of the contaminant-degrading microorganism(s) to compete for available resources with microorganisms already present in an aquifer or those that may contaminate a bioreactor. Though research has been performed studying the interaction of multiple species in batch and chemostat reactors, little work has been done looking at multi-species interactions in environments that more closely resemble field-scale applications. The research presented herein examined the interaction of Burkholderia cepacia PR1-pTOM(31c), an aerobic trichloroethylene (TCE)-degrading bacterium, with Klebsiella oxytoca, a facultative bacterium, in a flow-through porous media (PM) reactor. Growth characteristics and population distributions in PM were compared to previously reported values from batch and chemostat reactors. The faster growing organism in batch experiments (K. oxytoca) did not always have the greater population density in dual-species PM experiments. The biofilm population distribution was influenced by substrate concentration, with B. cepacia having a greater dual-species population density than K. oxytoca at a low (30 mg/L dissolved organic carbon [DOC]) substrate concentration and K. oxytoca having a greater population density at a high (700 mg/L DOC) substrate concentration. This change in species population distribution with change in substrate concentration, which was not observed in batch reactors, was also observed in chemostat reactors. Therefore, manipulation of substrate concentration enabled the control of species dominance to the advantage of the TCE degrading population in this dual-species PM system and may provide a mechanism to enhance bioremediation scenarios involving TCE or other contaminants of concern.