Evidence That High Activity of Vacuolar Invertase Is Required for Cotton Fiber and Arabidopsis Root Elongation through Osmotic Dependent and Independent Pathways,Respectively

Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due, in part, to the complexity of multicellular plant tissues. Here, we used cotton (Gossypium spp.) fibers, fast-growing single-celled seed trichomes, to address this issue. VIN activity in elongating fibers was approximately 4-6-fold higher than that in leaves, stems, and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Furthermore, a genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than a slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1, which displayed VIN sequence features and localized to the vacuole. Once introduced to Arabidopsis (Arabidopsis thaliana), GhVIN1 complemented the short-root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber, GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNA interference or overexpression constructs reduced or enhanced fiber elongation, respectively. Together, these analyses provide evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contributions of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively.Suc is the principal end product of photosynthesis in higher plants and the major carbohydrate translocated from source to sink tissues through phloem. Suc cleavage, serving as a starting point for various carbohydrate metabolic pathways, is catalyzed by Suc synthase (EC 2.4.1.13) and invertase (β-fructofuranosidase; EC 3.2.1.26). In contrast to the reversible reaction of Suc synthase, invertase irreversibly hydrolyzes Suc to Fru and Glc. This hydrolysis step is required for the development of many sink tissues and their responses to various stresses (Sturm, 1999; Weschke et al., 2003; Roitsch and González, 2004; Huang et al., 2007; Essmann et al., 2008; Jin et al., 2009; for a recent review, see Ruan et al., 2010).Based on their pH optimums and subcellular localizations, invertases are classified into three isoforms: a nonglycosylated cytosolic invertase (CIN), with an optimal pH of 7.0 to 7.8, and highly glycosylated acid invertases with an optimum pH of 3.5 to 5.5 either tightly bound to cell wall (CWIN) or appearing as a soluble form inside the vacuole (VIN; Roitsch and González, 2004). Mutational and transgenic studies have established the critical roles of CWIN in the development of seed (Cheng et al., 1996; Ruan et al., 2003), pollen (Roitsch et al., 2003), root (Tang et al., 1999), and leaf and fruit (Jin et al., 2009). By contrast, much less is known about the function of VIN or CIN (Ruan et al., 2010).High VIN expression or activity has been observed in a range of expanding tissues, including maize (Zea mays) ovaries (Andersen et al., 2002; McLaughlin and Boyer, 2004), grape (Vitis vinifera) berry (Davies and Robinson, 1996), carrot (Daucus carota) taproot (Tang et al., 1999), and sugar beet (Beta vulgaris) petioles (González et al., 2005). It is hypothesized that VIN may play a major role in plant cell expansion, a key step in plant cell development (González et al., 2005). However, progress in determining the roles of VIN in cell expansion suffers from several experimental limitations. Most notably, the multicellular nature of plant tissues makes it difficult to quantitatively evaluate the contribution of VIN in specific cell types. For example, decrease of VIN expression is associated with maize ovary abortion or reduction in its expansion (Andersen et al., 2002; McLaughlin and Boyer, 2004). The VIN gene Ivr2, however, is expressed in nucellus and vascular bundles of the pedicel deeply embedded within the pericarp (Andersen et al., 2002). This inherent anatomical feature makes it challenging to experimentally assess the role of invertase in these cells.In this context, developing cotton (Gossypium hirsutum) fiber offers a tractable experimental system to study the role of invertase in cell expansion for the following reasons. First, after initiation from the ovule epidermis at anthesis, the single-celled cotton fibers undergo rapid and synchronized unidirectional expansion to several centimeters long by approximately 18 d after anthesis (DAA; Ruan et al., 2001). Hence, a large quantity of homogenous single cells can be readily harvested for studying the control of cell expansion (Ruan, 2007). Second, compelling evidence has indicated a major role of osmotically active solutes in fiber elongation through the generation of cell turgor (Ruan et al., 2004). To this end, Suc moves into fibers symplasmically early in elongation (Ruan et al., 2001), and hexoses accumulated in the vacuole are major osmotically active solutes in the fiber sap (Dhindsa et al., 1975; Ruan et al., 1997), where VIN activity has been reported (Wäfler and Meier, 1994). These observations raise the possibility that VIN may be a central player in osmotically driven fiber expansion (Andersen et al., 2002; Ruan, 2005). Finally, elucidating the role of VIN in cotton fiber could help us not only better understand the control of rapid cell expansion but also identify novel ways to increase fiber length, a key quality and yield determinant of cotton, the most important textile crop worldwide (Ruan, 2005).This study aims to examine the role of VIN in cell expansion by using cotton fiber as a model, coupled with integrative analyses on elongating root of Arabidopsis (Arabidopsis thaliana). A combination of cellular, biochemical, and molecular genetic analyses show that (1) rapid fiber expansion requires high activity of VIN, which is probably exerted by the expression of GhVIN1, and (2) the impact on cotton fiber and Arabidopsis root elongation by VIN is most likely achieved through an osmotic dependent and independent manner, respectively.     

Global analysis of an epidemic model with nonmonotone incidence rate

In this paper we study an epidemic model with nonmonotonic incidence rate, which describes the psychological effect of certain serious diseases on the community when the number of infectives is getting larger. By carrying out a global analysis of the model and studying the stability of the disease-free equilibrium and the endemic equilibrium, we show that either the number of infective individuals tends to zero as time evolves or the disease persists.     

Clock controls timing of mouse pancreatic differentiation through regulation of Wnt- and Notch-based and cell division components

The oscillations of circadian genes control the daily circadian clock, regulating a diverse array of physiologies with the 24-hour light/dark cue across a wide variety of organisms. Here we first show that before embryonic circadian rhythms occur, the oscillation (nucleocytoplasmic shuttling) of core circadian gene Clock is tissue-specific and correlated with the state of differentiation during both early development and later pancreas organogenesis. Disruption of Clock as well as Timeless in the embryonic pancreas does not block pancreatic differentiation but alters the balance and maturity of endocrine and exocrine cells. Molecular analysis indicates that inhibition of Clock or Timeless expression disturbs not only cell cycle regulators, but also Wnt- and Notch-signaling components, whose oscillations establish the timing mechanism in somitogenesis. Thus, our results provide new insights about circadian genes' function in control of the timing of differentiation during embryonic development.     

Anti-atherosclerotic effects of sirolimus on human vascular smooth muscle cells

Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine IL-1 beta. We explored the effect of sirolimus on the lipid accumulation of VSMCs in the presence of IL-1 beta, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of sirolimus on the gene and protein expression of lipoprotein receptors and ATP binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and Western blotting, respectively. Furthermore, the effect of sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1 beta was also investigated using [(3)H] cholesterol efflux. Finally, we examined the effect of sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1 beta possibly due to the reduction of expression of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1 beta. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1 beta. We further confirmed that sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, tumor necrosis factor-alpha, IL-8, and monocyte chemoattractant protein-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of sirolimus on VSMCs.     

Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli

Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.     

Prediction of protein secondary structure content for the twilight zone sequences

Secondary protein structure carries information about local structural arrangements, which include three major conformations: alpha-helices, beta-strands, and coils. Significant majority of successful methods for prediction of the secondary structure is based on multiple sequence alignment. However, multiple alignment fails to provide accurate results when a sequence comes from the twilight zone, that is, it is characterized by low (<30%) homology. To this end, we propose a novel method for prediction of secondary structure content through comprehensive sequence representation, called PSSC-core. The method uses a multiple linear regression model and introduces a comprehensive feature-based sequence representation to predict amount of helices and strands for sequences from the twilight zone. The PSSC-core method was tested and compared with two other state-of-the-art prediction methods on a set of 2187 twilight zone sequences. The results indicate that our method provides better predictions for both helix and strand content. The PSSC-core is shown to provide statistically significantly better results when compared with the competing methods, reducing the prediction error by 5-7% for helix and 7-9% for strand content predictions. The proposed feature-based sequence representation uses a comprehensive set of physicochemical properties that are custom-designed for each of the helix and strand content predictions. It includes composition and composition moment vectors, frequency of tetra-peptides associated with helical and strand conformations, various property-based groups like exchange groups, chemical groups of the side chains and hydrophobic group, auto-correlations based on hydrophobicity, side-chain masses, hydropathy, and conformational patterns for beta-sheets. The PSSC-core method provides an alternative for predicting the secondary structure content that can be used to validate and constrain results of other structure prediction methods. At the same time, it also provides useful insight into design of successful protein sequence representations that can be used in developing new methods related to prediction of different aspects of the secondary protein structure.     

The different roles of cyclinD1-CDK4 in STP and mGluR-LTD during the postnatal development in mice hippocampus area CA1

Background

The extraembryonic tissues, visceral endoderm (VE) and extraembryonic ectoderm (ExE) are known to be important for the induction of primordial germ cells (PGCs) in mice via activation of the bone morphogenetic protein (BMP) signalling pathway. We investigated whether the VE and ExE have a direct role in the specification of PGCs, or in an earlier event, namely the induction of the PGC precursors in the proximal posterior epiblast cells.

Results

We cultured embryonic day (E) 5.75 to E7.0 mouse embryos in an explant-assay with or without extraembryonic tissues. The reconstituted pieces of embryonic and extraembryonic tissues were assessed for the formation of both PGC precursors and specified PGCs. For this, Blimp1:gfp and Stella:gfp transgenic mouse lines were used to distinguish between PGC precursors and specified PGC, respectively. We observed that the VE regulates formation of an appropriate number of PGC precursors between E6.25–E7.25, but it is not essential for the subsequent specification of PGCs from the precursor cells. Furthermore, we show that the ExE has a different role from that of the VE, which is to restrict localization of PGC precursors to the posterior part of the embryo.

Conclusion

We show that the VE and ExE have distinct roles in the induction of PGC precursors, namely the formation of a normal number of PGC precursors, and their appropriate localization during early development. However, these tissues do not have a direct role during the final stages of specification of the founder population of PGCs.     

Prediction of flexible/rigid regions from protein sequences using k-spaced amino acid pairs

Background

Traditionally, it is believed that the native structure of a protein corresponds to a global minimum of its free energy. However, with the growing number of known tertiary (3D) protein structures, researchers have discovered that some proteins can alter their structures in response to a change in their surroundings or with the help of other proteins or ligands. Such structural shifts play a crucial role with respect to the protein function. To this end, we propose a machine learning method for the prediction of the flexible/rigid regions of proteins (referred to as FlexRP); the method is based on a novel sequence representation and feature selection. Knowledge of the flexible/rigid regions may provide insights into the protein folding process and the 3D structure prediction.

Results

The flexible/rigid regions were defined based on a dataset, which includes protein sequences that have multiple experimental structures, and which was previously used to study the structural conservation of proteins. Sequences drawn from this dataset were represented based on feature sets that were proposed in prior research, such as PSI-BLAST profiles, composition vector and binary sequence encoding, and a newly proposed representation based on frequencies of k-spaced amino acid pairs. These representations were processed by feature selection to reduce the dimensionality. Several machine learning methods for the prediction of flexible/rigid regions and two recently proposed methods for the prediction of conformational changes and unstructured regions were compared with the proposed method. The FlexRP method, which applies Logistic Regression and collocation-based representation with 95 features, obtained 79.5% accuracy. The two runner-up methods, which apply the same sequence representation and Support Vector Machines (SVM) and Naïve Bayes classifiers, obtained 79.2% and 78.4% accuracy, respectively. The remaining considered methods are characterized by accuracies below 70%. Finally, the Naïve Bayes method is shown to provide the highest sensitivity for the prediction of flexible regions, while FlexRP and SVM give the highest sensitivity for rigid regions.

Conclusion

A new sequence representation that uses k-spaced amino acid pairs is shown to be the most efficient in the prediction of the flexible/rigid regions of protein sequences. The proposed FlexRP method provides the highest prediction accuracy of about 80%. The experimental tests show that the FlexRP and SVM methods achieved high overall accuracy and the highest sensitivity for rigid regions, while the best quality of the predictions for flexible regions is achieved by the Naïve Bayes method.