Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine,verticinone and isoverticine in rat plasma

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC–LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 μm particle size (Agilent Zorbax StableBond-C₁₈, 4.6 mm × 50 mm, 1.8 μm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic–lipophilic balanced copolymer (2.1 mm × 20 mm, 25 μm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC–MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R > 0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.     

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.     

Telocytes accompanying cardiomyocyte in primary culture: two- and three-dimensional culture environment

Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra-digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two- and three-dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two-dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three-dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c-kit or CD34 immuno-positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three-dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non-muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.     

Synthesis of novel ligustrazine derivatives as NA+/H+ exchange inhibitors

A novel series of 3,5,6‐trimethylpyrazine‐2‐methoxy (or methylamino) substituted benzoyl‐guanidine derivatives were designed and synthesized as Na⁺/H⁺ exchange (NHE) inhibitors. In this study, compounds with electron‐withdrawing substituents on the benzene ring seemed to improve NHE‐1 inhibitory activities. Compounds 6d, 6k , and 6l were found to be potent inhibitors of NHE‐1 (IC₅₀=3.0±1.6, 3.0±1.4, and 1.6±0.4 nmol/l, resp.). Furthermore, they showed a remarkable reduction of infarct size in the rat myocardial infarction model in vivo.     

Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

An expression‐uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP‐bait protein from the target cells. Here, the TAP‐tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue‐specific binding partners of the protein kinase A catalytic subunit Cβ1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor‐dependent interaction of Cβ1 with the cell cycle and apoptosis regulatory protein‐1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell‐ and tissue‐specific protein complexes.     

Unique biomechanical interactions between myeloma cells and bone marrow stroma cells

We observed that BMSCs (bone marrow stromal cells) from myeloma patients (myeloma BMSCs) were significantly stiffer than control BMSCs using a cytocompression device. The stiffness of myeloma BMSCs and control BMSCs was further increased upon priming by myeloma cells. Additionally, myeloma cells became stiffer when primed by myeloma BMSCs. The focal adhesion kinase activity of myeloma cells was increased when cells were on stiffer collagen gels and on myeloma BMSCs. This change in myeloma stiffness is associated with increased colony formation of myeloma cells and FAK activation when co-cultured with stiffer myeloma BMSCs or stiffer collagen. Additionally, stem cells of RPMI8226 cells became stiffer after priming by myeloma BMSCs, with concomitant increases of stem cell colony formation. These results suggest the presence of a mechanotransduction loop between myeloma cells and myeloma BMSCs to increase the stiffness of both types of cells via FAK activation. The increase of stiffness may in turn support the growth of myeloma cells and myeloma stem cells.     

Forced flowering of pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> cv. Tainon 17) in response to cold stress,ethephon and calcium carbide with or without activated charcoal

Ethylene, a gaseous plant hormone, is responsible for the initiation of reproductive development in pineapple. Reproductive development can be forced in pineapple (Ananas comosus var. comosus) throughout the year with ethylene. Inhibition of natural flowering initiation with aviglycine [(S)-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride], an inhibitor of ethylene biosynthesis, provides evidence that reproductive development in response to cold stress and short daylength is also in response to ethylene production. We studied the effect of cold treatment of pineapple on ethylene production and flower induction by applying a short-term cold stress to stem apices. Shoot apices of pineapple treated with ice crystals also produced twice as much ethylene as did those of control plants and significantly more than was produced by “D” leaf basal tissue. Moreover, pineapple plants treated four times with ice crystals or ice water were induced to flower under field conditions and the forcing efficiency, as evaluated by the percentages of inflorescence emergence and fruit harvest, was comparable to forcing with calcium carbide (CaC₂) and ethephon. In another field experiment two applications of a 1.0% solution of CaC₂ or 0.15% ethephon applied at 48 h intervals was sufficient to force reproductive development of ‘Tainon 17’. Furthermore, 0.5 or 1.0% solutions of CaC₂ supplemented with 0.5% activated charcoal (AC) significantly improved the forcing effectiveness of CaC₂. This could/would make it possible to reduce the number or concentration, or both, of CaC₂ required to effect forcing in pineapple.     

Pellionia ronganensis sp. nov. (Urticaceae) from Guangxi, China

Pellionia ronganensis sp. nov. (Urticaceae) is described and illustrated. Pellionia ronganensis resembles P. incisoserrata (H. Schroter) W. T. Wang, but is distinguished by leaves with obtuse theef, larger (6–10 mm long), linear stipules and tuberculate achenes.