A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l^-1 cellulase Onozuka R10, 1 g l^-1 Pectolyase Y-23 and 10 g l^-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×10⁴ protoplasts ml^-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l^-1 naphthaleneacetic acid and 1 mg l^-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×10⁴ protoplasts ml^-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l^-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l^-1 naphthaleneacetic acid, 0.05 mg l^-1 indole-3-butyric acid and 0.1 mg l^-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.     

The preparation in vitro of chrysanthemum for transplantation to soil

Plantlets of Dendranthema grandiflora Pennine Reel were grown from nodal sections on Sorbarods saturated with liquid medium containing 0–3 mg 1^–1 of various growth retardants. After 4 weeks they were transferred to compost and maintained at a relative humidity of 42% at 27.5°C. Wilting was assessed over a period of 3 h. Plantlets treated with paclobutrazol, flurprimidol, triapenthenol, chlorphonium chloride, uniconazol and ancymidol showed dose-related reductions in wilting up to a concentration of 3 mg 1^–1. Responses to tetcyclacis and mepiquat chloride were weaker, and no responses to chlormequat chloride, BTS 44584 or diaminozide could be detected. These observations are compatible with an hypothesis that resistance to wilting derives from inhibited synthesis of gibberellins.     

Sulfate anion free radical formation by the peroxidation of (Bi)sulfite and its reaction with hydroxyl radical scavengers

The one-electron oxidation of (bi)sulfite is catalyzed by peroxidases to yield the sulfur trioxide radical anion (SO3-), a predominantly sulfur-centered radical as shown by studies with 33S-labeled (bi)sulfite. This radical reacts with molecular oxygen to form a peroxyl radical. The subsequent reaction of this peroxyl radical with (bi)sulfite has been proposed to form the sulfate anion radical, which is nearly as strong an oxidant as the hydroxyl radical. We used the spin trapping electron spin resonance technique to provide for the first time direct evidence for sulfate anion radical formation during (bi)sulfite peroxidation. The sulfate anion radical is known to react with many compounds more commonly thought of as hydroxyl radical scavengers such as formate and ethanol. Free radicals derived from these scavengers are trapped in systems where (bi)sulfite peroxidation has been inhibited by these scavengers.     

An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase

The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.     

Free radical metabolites of L-cysteine oxidation

The oxidation of L-cysteine by horseradish peroxidase in the presence of oxygen forms a thiyl free radical as demonstrated with the spin-trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. Cysteine sulfinic acid, a cysteine metabolite, is a poorer substrate for horseradish peroxidase than cysteine and is oxidized to form both sulfur-centered and carbon-centered free radicals.     

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 10⁶ cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 10⁸ cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10⁸ cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 10⁸ cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO₂ level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10⁸ cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013     

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

The development of phenanthrene catabolism in soil amended with transformer oil

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants frequently associated with light non-aqueous-phase liquids (LNAPLs) in soil. Microbial degradation comprises a major loss process for PAHs in the environment. Various laboratory studies, using known degraders, have shown reduced or enhanced mineralisation of PAHs when dissolved in different LNAPLs. Effects due to the presence of LNAPLs on indigenous micro-organisms, however, are not fully understood. A pristine pasture soil was spiked with [14C]phenanthrene and transformer oil to 0, 0.01 and 0.1%, and incubated for 180 days. The catabolic potential of the soil towards phenanthrene was assessed periodically during ageing. The extent of the lag phase (prior to >5% mineralisation), maximum rates and overall extents of mineralisation observed during the course of a 14-day bioassay appeared to be dependent upon phenanthrene concentration, the presence of transformer oil, and soil-contaminant contact time. Putatively, transformer oil enhanced acclimation and facilitated the development of measurable catabolic activity towards phenanthrene in a previously uncontaminated pasture soil. Exact mechanisms for the observed enhancement, longer-term fate/degradation of the oil and residual phenanthrene, and effects of the presence of the oil on the indigenous microbes over extended time frames warrant further investigation.     

Vaccine-induced tumor-specific immunity despite severe B-cell depletion in mantle cell lymphoma

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell-dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody-based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4(+) and CD8(+) antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.     

Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.