Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies

Sec14p promotes the energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between lipid bilayers in vitro and represents the major PtdIns/PtdCho transfer protein in the budding yeast Saccharomyces cerevisiae. Herein, we employ multi-frequency high-field electron paramagnetic resonance (EPR) to analyze the electrostatic and hydrogen-bonding microenvironments for series of doxyl-labeled PtdCho molecules bound by Sec14p in a soluble protein-PtdCho complex. A structurally similar compound, 5-doxyl stearic acid dissolved in a series of solvents, was used for experimental calibration. The experiments yielded two-component rigid limit 130- and 220-GHz EPR spectra with excellent resolution in the gx region. Those components were assigned to hydrogen-bonded and nonhydrogen-bonded nitroxide species. Partially resolved 130-GHz EPR spectra from n-doxyl-PtdCho bound to Sec14p were analyzed using this two-component model and allowed quantification of two parameters. First, the fraction of hydrogen-bonded nitroxide species for each n-doxyl-PtdCho was calculated. Second, the proticity profile along the phospholipid-binding cavity of Sec14p was characterized. The data suggest the polarity gradient inside the Sec14p cavity is a significant contributor to the driving molecular forces for extracting a phospholipid from the bilayer. Finally, the enhanced g-factor resolution of EPR at 130 and 220 GHz provides researchers with a spectroscopic tool to deconvolute two major contributions to the x-component of the nitroxide g-matrix: hydrogen-bond formation and local electrostatic effects.     

Focus on function: single molecule RNA enzymology

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.     

Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression

Agrobacterium-mediated transient expression may be regarded as a promising method for inexpensive large-scale production of recombinant proteins. We optimized the protocol of transient expression in Nicotiana benthamiana and compared six Australian species of Nicotiana as hosts for transient expression. The transient expression of GFP under 35S CaMV promoter was observed in all species tested, although the GFP content in leaves of N. benthamiana, N. exigua, and N. excelsior was significantly higher (3.8, 3.7, and 2.0% TSP, respectively). Usage of viral-based expression system resulted in considerable increase of GFP accumulation in N. excelsior and N. benthamiana (63.5 and 16.2% TSP, respectively). We displayed that N. excelsior has the best characteristics in regard to biomass yield as well as GFP accumulation level for both types of the expression cassettes tested.     

Comparison of characteristics of cultured limbal cells on denuded amniotic membrane and fresh conjunctival, limbal and corneal tissues

This study aimed to evaluate proposed molecular markers related to eye limbal stem cells (SC) and to identify novel associated genes. The expression of a set of genes potentially involved in stemness was assessed in freshly prepared limbal, corneal and conjunctival tissues. PAX6, AC133, K12 and OCT4 were detected in all the tissues and p63(+)/K3(-)/K12(+)/Nodal(+)/Cx43(+) were expressed in conjunctival, p63(-)/K3(+)/K12(+)/Nodal(-)/Cx43(+) in corneal, and p63(+)/K3(-)/K12(-)/Nodal(-)/Cx43(-) in limbal tissues. Limbal explants were cultured on human amniotic membrane for 21 days. The cells expressed p63 but not K3, K12, Nodal and Cx43, however, the expression of K3, K12 and Cx43 was detected, and p63 and the high BrdU-labeling index decreased with more culture. Ultrastructure analysis of the cultured cells showed typically immature organization of intracellular organelles and architecture. Our data suggest that limbal, corneal and conjunctival tissues are heterogeneous with some progenitors. Also, the expression of traditional SC markers may not be a reliable indicator of limbal SC and there is an increasing need to determine factor(s) involved in their stemness.     

Apoptosis regulates notochord development in Xenopus

The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed.     

The significance of multiple mating in the social wasp Vespula maculifrons

The evolution of the complex societies displayed by social insects depended partly on high relatedness among interacting group members. Therefore, behaviors that depress group relatedness, such as multiple mating by reproductive females (polyandry), are unexpected in social insects. Nevertheless, the queens of several social insect species mate multiply, suggesting that polyandry provides some benefits that counteract the costs. However, few studies have obtained evidence for links between rates of polyandry and fitness in naturally occurring social insect populations. We investigated if polyandry was beneficial in the social wasp Vespula maculifrons. We used genetic markers to estimate queen mate number in V. maculifrons colonies and assessed colony fitness by counting the number of cells that colonies produced. Our results indicated that queen mate number was directly, strongly, and significantly correlated with the number of queen cells produced by colonies. Because V. maculifrons queens are necessarily reared in queen cells, our results demonstrate that high levels of polyandry are associated with colonies capable of producing many new queens. These data are consistent with the explanation that polyandry is adaptive in V. maculifrons because it provides a fitness advantage to queens. Our research may provide a rare example of an association between polyandry and fitness in a natural social insect population and help explain why queens in this taxon mate multiply.