Garden cress (Lepidium sativum L.) is an edible, underutilised herb, grown mainly for its seeds in India. Physicochemical properties, minor components (unsaponifiable matter, tocopherols, carotenoids), fatty acid composition and storage stability of garden cress seed oil (GCO) were studied. Cold press, solvent and supercritical CO 2 extraction methods were employed to extract the oil. The total oil content of garden cress (GC) seeds was 21.54, 18.15 and 12.60% respectively by solvent, supercritical CO 2 and cold press methods. The physical properties of GCO extracted by the above methods were similar in terms of refractive index, specific gravity and viscosity. However, cold pressed oil showed low PV and FFA compared to the oil extracted by other methods. a-Linolenic acid (34%) was the major fatty acid in GCO followed by oleic (22%), linoleic (11.8%), eicosanoic (12%), palmitic (10.1%) erucic (4.4%), arachidic (3.4%) and stearic acids (2.9%). Oleic acid (39.9%) and a-linolenic acid (42.1%) were the predominant fatty acids at the sn-2 position. The total tocopherol and carotenoid content of GCO was 327.42 and 1.0 lmol/100 g oil, respectively. The oil was stable up to 4 months at 4°C. Tocopherol and BHT offered the least protection, while ascorbyl palmitate (200 ppm) offered the maximum protection to the oil, when subjected to the accelerated oxidative stability test. Thus GCO can be considered as a fairly stable oil with a high content of a-linolenic acid.
The TREX (transcription/export) complex has been conserved throughout evolution from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. The TREX complex in mammals and Drosophila is composed of the THO subcomplex (THOC1, THOC2, THOC5, THOC6, and THOC7), THOC3, UAP56, and Aly/THOC4. In human and Drosophila, various studies have shown that THO is required for the export of heat shock mRNAs, but nothing is known about other mRNAs. Our previous study using conditional THOC5 (or FMIP ) knockout mice revealed that the presence of THOC5 is critical in hematopoietic cells but not for terminally differentiated cells. In this study, we describe the establishment of a mouse embryo fibroblast cell line (MEF), THOC5 flox/flox. Four days after infection of MEF THOC5 flox/flox with adenovirus carrying Cre-recombinase gene (Ad-GFP-Cre), THOC5 is down-regulated >95% at the protein level, and cell growth is strongly suppressed. Transcriptome analysis using cytoplasmic RNA isolated from cells lacking functional THOC5 reveals that only 2.9% of all genes were down-regulated more than twofold. Although we examined these genes in fibroblasts, one-fifth of all downregulated genes (including HoxB3 and polycomb CBX2) are known to play a key role in hematopoietic development. We further identified 10 genes that are spliced but not exported to the cytoplasm in the absence of THOC5. These mRNAs were copurified with THOC5. Furthermore, Hsp70 mRNA was exported in the absence of THOC5 at 37°C, but not under heat shock condition (42°C), suggesting that THOC5 may be required for mRNA export under stress and/or upon signaling-induced conditions.
During animal development, cells need to sense and adapt to mechanical forces from their environment. Ultimately, these forces are transduced through the actomyosin cortex. How the cortex simultaneously responds to and creates forces during cytokinesis is not well understood. Here we show that, under mechanical stress, cortical actomyosin flow can switch polarization during cytokinesis in the C. elegans embryo. In unstressed embryos, longitudinal cortical flow contributes to contractile ring formation, while rotational cortical flow is additionally induced in uniaxially loaded embryos, i.e. embryos compressed between two plates. Rotational flow depends on astral microtubule signals and is required for the redistribution of the actomyosin cortex in loaded embryos. Rupture of longitudinally aligned cortical fibers during cortex rotation releases tension, initiates orthogonal longitudinal flow and, thereby, contributes to furrowing in loaded embryos. Moreover, actomyosin regulators involved in RhoA regulation, cortical polarity and chirality are all required for rotational flow, and become essential for cytokinesis under mechanical stress. In sum, our findings extend the current framework of mechanical stress response during cell division and show scaling of orthogonal cortical flows to the amount of mechanical stress.
Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics.
Formins are multi domain proteins present ubiquitously in all eukaryotes from lower fungi to higher vertebrates. Formins are characterized by the presence of formin homology domain-2 (FH2) and formin homology domain-1 (FH1). There are fifteen different formins present in mouse and human. Among these metazoan formins, Delphilin is a unique formin having two PDZ domains at the N-terminus and FH1, FH2 domain at the C-terminus respectively. In this study we observed that Delphilin binds to actin filaments, and Delphilin inhibits actin filament elongation like barbed end capping protein CapZ. In vitro, Delphilin stabilized actin filaments by inhibiting actin filament depolymerisation. Therefore, our study demonstrates Delphilin as an actin-filament capping protein.
Nipah is an emerging paramyxovirus that is of serious concern to human health. It invades host cells using two of its membrane proteins-G and F. G binds to host ephrins and this stimulates G to activate F. Upon activation, F mediates virus-host membrane fusion. Here we focus on mechanisms that underlie the stimulation of G by ephrins. Experiments show that G interacts with ephrin and F through separate sites located on two different domains, the receptor binding domain (RBD) and the F activation domain (FAD). No models explain this allosteric coupling. In fact, the analogous mechanisms in other paramyxoviruses also remain undetermined. The structural organization of G is such that allosteric coupling must involve at least one of the two interfaces-the RBD-FAD interface and/or the RBD-RBD interface. Here we examine using molecular dynamics the effect of ephrin binding on the RBD-RBD interface. We find that despite inducing small changes in individual RBDs, ephrin reorients the RBD-RBD interface extensively, and in a manner that will enhance solvent exposure of the FAD. While this finding supports a proposed model of G stimulation, we also find from additional simulations that ephrin induces a similar RBD-RBD reorientation in a stimulation-deficient G mutant, V 209 VG / AAA. Together, our simulations suggest that while inter-RBD reorientation may be important, it is not, by itself, a sufficient condition for G stimulation. Additionally, we find that the mutation affects the conformational ensemble of RBD globally, including the RBD-FAD interface, suggesting the latter's role in G stimulation. Because ephrin induces small changes in individual RBDs, a proper analysis of conformational ensembles required that they are compared directly-we employ a method we developed recently, which we now release at SimTK, and show that it also performs excellently for non-Gaussian distributions.
Formation of the anteroposterior and dorsoventral body axis in Caenorhabditis elegans depends on cortical flows and advection of polarity determinants. The role of this patterning mechanism in tissue polarization after formation of cell-cell contacts is not fully understood. Here, we demonstrate that planar asymmetries are established during left-right symmetry breaking: Centripetal cortical flows asymmetrically and differentially advect anterior polarity determinants (aPARs) from contacts to the medial cortex, resulting in their unmixing from apical myosin. Contact localization and advection of PAR-6 requires balanced CDC-42 activation, while asymmetric retention and advection of PAR-3 can occur independently of PAR-6. Concurrent asymmetric retention of PAR-3, E-cadherin/HMR-1 and opposing retention of antagonistic CDC-42 and Wnt pathway components leads to planar asymmetries. The most obvious mark of planar asymmetry, retention of PAR-3 at a single cell-cell contact, is required for proper cytokinetic cell intercalation. Hence, our data uncover how planar polarity is established in a system without the canonical planar cell polarity pathway through planar asymmetric retention of aPARs.
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