Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells

Vascular endothelial dysfunction is thought to play a prominent role in systemic anthrax pathogenesis. We examined the effect of anthrax lethal toxin (LTx), a key virulence factor of Bacillus anthracis, on the expression of vascular cell adhesion molecule-1 (VCAM-1) on normal and cytokine-stimulated human lung microvascular endothelial cells. Confluent endothelial monolayers were treated with lethal factor (LF), protective antigen (PA), or both (LTx) in the presence or absence of tumor necrosis factor-alpha (TNFalpha). LTx enhanced cytokine-induced VCAM-1 expression and monocyte adhesion. LTx alone had no effect on VCAM-1 expression. LF, PA or the combination of a catalytically inactive mutant LF and PA failed to enhance cytokine-induced VCAM-1 expression. Treatment with inhibitors of mitogen-activated protein kinase kinases (MEKs) and mitogen-activated protein kinases did not reproduce the VCAM-1 enhancement effect of LTx, a known MEK metalloprotease, suggesting LTx-mediated MEK cleavage may not be a contributing factor.     

The Wound-Inducible Lls1 Gene from Maize is an Orthologue of the Arabidopsis Acd1 Gene,and the LLS1 Protein is Present in Non-Photosynthetic Tissues

Previous studies indicated that the lethal leaf spot 1 lesion mimic locus of maize ( ZmLls1 ) encodes a novel cell protective function in plants. Here we show that the accelerated cell death 1 ( acd1 ) locus of Arabidopsis thaliana corresponds to gene At3g44880 on chromosome 3. Proof that the Acd1 gene is an orthologue of ZmLls1 is provided by in vivo complementation of the acd1 mutant by the ZmLls1 gene. The Atlls1 lesion mimic phenotype was delayed in a chlorophyll a oxygenase (CAO) mutant chlorina1 background which is deficient in chlorophyll b synthesis. The interpretation that the cell protective function of LLS1 is linked with the removal of a phototoxic chlorophyll intermediate is supported by the recent report that the maize Lls1 gene encodes pheophorbide a oxygenase (PaO). Western blot analysis demonstrates that the LLS1 protein is present constitutively in all photosynthetic plant tissues. A transient increase in Lls1 gene expression by about 50-fold upon physical wounding of maize leaves indicates that the function of Lls1 is regulated in response to stress. We show that the LLS1 protein is also present at low levels in non-photosynthetic tissues including etiolated leaves suggesting that the ability to degrade chlorophyll exists in a standby mode in plant cells.     

The variability of Vipera ammodytes ammodytes venoms from Croatia--biochemical properties and biological activity

Vipera ammodytes ammodytes venom has been used for many years in Croatia for immunization of horses and production of specific therapeutic anti-venoms. The neutralizing effectiveness of anti-venoms is directly dependent on the properties of the snake venom used for immunization. Therefore, appropriate characterization of the whole venom is necessary prior to use in the immunization procedure. In the course of such analyses, the variability in biochemical properties and biological activity was observed in venoms collected from snakes originating from different parts of Croatia. The venom pools also differed with respect to time of snake collection (1992-2003). Analyses of three samples of whole venom pools were carried out revealing differences in lethal activity (LD50), minimum haemorrhagic dose (MHD), minimum necrotizing dose (MND), phospholipase A2 activity and in anticomplementary activity. SDS-PAGE electrophoretic patterns were similar, but not identical, for all tested venom pools with respect to the number of protein bands detected, but intensity of particular components differed. Preliminary immunogenicity testing in terms of determination of specific antibodies revealed similar immunogenicity and high cross-reactivity for three samples tested.     

核型多角体病毒对宿主昆虫致死速率与温度关系的数学模拟

提出了核型多角体病毒（ＮＰＶ）对宿主幼虫平均致死速率〔Ｖ（Ｔ）〕与温度关系的数学模型,该模型能很好地描述茶尺蠖ＮＰＶ感染各龄初幼虫后,平均２死速率与温度的民适于描美洲棉铃虫、大蜡暝和黎豆夜蛾等ＮＰＶ对各自宿主幼虫的平均致死速率与温度的关系。     

Polarized Organization of the Cytoskeleton: Regulation by Cell Polarity Proteins

Polarity is one of the fundamental properties displayed by living organisms. In metazoans, cell polarity governs developmental processes and plays an essential role during maintenance of forms of tissues as well as their functions. The mechanisms of establishment and maintenance of cell polarity have been investigated extensively in the last two decades. This has resulted in identification of “core cell polarity modules” that control anterior–posterior, front–rear and apical–basal polarity across various cell types. Here, we review how these polarity modules interact closely with the cytoskeleton during establishment and maintenance of cytoskeletal polarity. We further suggest that reciprocal interactions between cell polarity modules and the cytoskeleton consolidate the initial weaker polarity, arising from an external cue, into a committed polarized system.     

A new method for hybrid seed production based on cytoplasmic male sterility combined with a lethal gene and a female sterile pollenizer in Capsicum annuum L.

Summary A new improved method for hybrid seed production was successfully tested. This method is based on using a cytoplasmic male sterile line possessing a lethal gene with action that can be easily inhibited and a female sterile pollenizer. The lethal gene ensures 100% purity of the F₁ crop. The female sterile pollenizer provides a permanent abundant flowering with excess of pollen grains that leads to increased hybrid seed production without additional labour expenses. The described scheme is applicable for other crops as well.     

Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.     

Boolean network-based analysis of the apoptosis network: Irreversible apoptosis and stable surviving

To understand the design principles of the molecular interaction network associated with the irreversibility of cell apoptosis and the stability of cell surviving, we constructed a Boolean network integrating both the intrinsic and extrinsic pro-apoptotic pathways with pro-survival signal transduction pathways. We performed statistical analyses of the dependences of cell fate on initial states and on input signals. The analyses reproduced the well-known pro- and anti-apoptotic effects of key external signals and network components. We found that the external GF signal by itself did not change the apoptotic ratio from randomly chosen initial states when there is no external TNF signal, but can significantly offset apoptosis induced by the TNF signal. While a complete model produces the expected irreversibility of the apoptosis process, alternative models missing one or more of four selected inter-component connections indicate that the feedback loops directly involving the caspase 3 are essential for maintaining irreversibility of apoptosis. The feedback loops involving P53 showed compensating effects when those involving caspase 3 have been removed. The GF signal significantly increases the stability of the surviving states of the network. The apoptosis network seems to use different modules by design to control the irreversibility of the apoptosis process and the stability of the surviving states. Such a design may accommodate the needed plasticity for the network to adapt to different cellular environments: depending on the strength of external pro-surviving signals, apoptosis can be induced either easily or difficultly by pro-apoptotic signal of varying strengths, but proceed with invariable irreversibility.     

Cold tolerance of the overwintering larval instars of light brown apple moth Epiphyas postvittana

The light brown apple moth, Epiphyas postvittana, a leafroller native to southeastern Australia was discovered in California in 2006. The highly polyphagous nature of this pest adds to the importance of being able to predict the potential distribution of this invader across the North American continent. The spread of ectothermic species that lack winter diapause, such as E. postvittana, can be limited by their ability to tolerate cold temperature extremes. In this study we examined the cold hardiness of 4th to 6th instar E. postvittana, the only life stages known to overwinter in California, through a combination of supercooling point (SCP) and mortality at low temperatures. Our results showed that the mean SCP for E. postvittana ranged from −14.1 °C for 6th instars to −16.0 °C for 4th instars. Lethal time leading to 50% mortality (LT₅₀) for the three instars combined were 2.5 h at −10.5 °C, 41 h at −6.5 °C and 198 h at −0.9 °C. At 3 °C, the LT₅₀ of 4th instars was significantly lower at 775 h than that for 5th and 6th instars combined at 1029 h. The cold hardiness characteristics of later-instar E. postvittana larvae were comparable to those of pink bollworm, Pectinophora gossypiella, a diapausing invasive with a geographic distribution restricted to southern California. Slightly greater cold hardiness is shown by the indigenous non-diapausing leafroller Argyrotaenia franciscana, which is restricted to the Pacific Coast of North America. We therefore conclude that the moderate cold hardiness of E. postvittana will substantially limit its spread into northern temperate regions of North America.     

Par-4 mediated Smad4 induction in PDAC cells restores canonical TGF-β/ Smad4 axis driving the cells towards lethal EMT

Deregulation of TGF-β signaling is intricately engrossed in the pathophysiology of pancreatic adenocarcinomas (PDACs). The role of TGF-β all through pancreatic cancer initiation and progression is multifarious and somewhat paradoxical. TGF-β plays a tumor suppressive role in early-stage pancreatic cancer by promoting apoptosis and inhibiting epithelial cell cycle progression, but incites tumor promotion in late-stage by modulating genomic instability, neo-angiogenesis, immune evasion, cell motility, and metastasis. Here, we provide evidences that Par-4 acts as one of the vital mediators to regulate TGF-β/Smad4 pathway, wherein, Par-4 induction/over-expression induced EMT which was later culminated in to apoptosis in presence of TGF-β via positive regulation of Smad4. Intriguingly, Par-4^−/− cells were devoid of significant Smad4 induction compared to Par-4^+/+ cells in presence of TGF-β and ectopic Par-4 steadily augmented Smad4 expression by restoring TGF-β/Smad4 axis in Panc-1 cells. Further, our FACS and western blotting results unveiled that Par-4 dragged the PDAC cells to G₁ arrest in presence of TGF-β byelevating p21 and p27 levels while attenuating Cyclin E and A levels and augmenting caspase 3 cleavage triggering lethal EMT. Through restoration of Smad4, we further establish that in BxPC3 cell line (Smad4^-/-), Smad4 is essential for Par-4 to indulge TGF-β dependent lethal EMT program. The mechanistic relevance of Par-4 mediated Smad4 activation was additionally validated by co-immunoprecipitation wherein disruption of NM23H1-STRAP interaction by Par-4 rescues TGF-β/Smad4 pathway in PDAC and mediates the tumor suppressive role of TGF-β, therefore serving as a vital cog to restore the apoptotic functions of TGF-β pathway.