PA,a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.¹ PA was shown to inhibit (1) CTR1''s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.Key words: lipid signaling, phosphatidic acid, ethylene, constitutive triple response 1, plant stress signaling, protein kinase, phospholipase D     

Three‐dimensional cell culture model utilization in cancer stem cell research

Three‐dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.     

The utility of the morphological variation of pollen for resolving the evolutionary history of Billia (subfam. Hippocastanoideae,Sapindaceae)

In this study, we examined the utility of pollen morphology for resolving questions about the evolutionary history of Billia, which is a poorly known genus of Neotropical trees. Billia has been traditionally circumscribed with two species and treated as sister to Aesculus L. However, the number of species in Billia is uncertain, because the genus exhibits abundant morphological diversity but little discontinuous variation. Therefore, Billia may be monotypic and highly polymorphic, or it may have two species with blurred boundaries due to incipient speciation and/or hybridization. Moreover, one recent molecular phylogenetic study shows Billia nested withinAesculus. Our work sought to address the following questions: (i) Are there discontinuities in the pollen of Billia that may suggest species boundaries? (ii) Does the pollen of Billia show evidence for inter-specific hybridization? (iii) Do the exine morphology and size of pollen in Billia differ from those in Aesculus? Our results from scanning electron microscopy showed that pollen exine morphology is not taxonomically informative in Billia but that there are significant differences in pollen size between red- and white-flowered individuals. Thus, our pollen data support the utility of flower color in Billia for species delimitation. Our assessments of pollen viability do not support hybridization in the genus, but cannot be used to rule it out. Finally, pollen exine morphology may lend some support to an evolutionary origin ofBillia within eastern North American Aesculus. In contrast, data on pollen size suggest that Billia may belong in a topological position outside of Aesculus.     

Evolutionarily stable allocation to vegetative and sexual reproduction in plants

I apply the resource allocation approach to construct an ESS model aimed at predicting the rate of vegetative and sexual reproduction in plants. The model provides an integrated explanation of why the mode of reproduction should vary between populations and species, and what factors should affect the trade-off between ramet and seed production. It follows that seed production, usually considered to be less effective and more costly than ramet production, may be maintained in the population. The production of ramets should be favored when their recruitment rate is high or the costs are low. The intensity of vegetative reproduction is also related to conditions which affect seed production directly. The rate of ramet production should be inversely correlated with factors favoring sexual reproduction, such as lower cost of seeds or high opportunity for colonization of new patches, etc. Under such conditions it is beneficial to allocate more resources available for reproduction into seeds, at the expense of ramet production. The model emphasizes the need to consider both modes of plant reproduction whenever one of them is theoretically or empirically studied.     

A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments

Summary A general approach for assigning the resonances of uniformly ¹⁵N- and ¹³C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The ¹H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D ¹⁵N-edited ¹H, ¹H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete ¹³C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the ¹H, ¹³C, and ¹⁵N resonances of the FK506 binding protein denatured in 6.3 M urea.     

1H, 13C,and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin

The ¹H, ¹³C, and ¹⁵N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-¹⁵N] FKBP/ascomycin and [U-¹⁵N, ¹³C] FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, ³J coupling constants, amide exchange data, and the differences between the C^α and C^β chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution. © 1993 John Wiley & Sons, Inc.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.     

NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.