Solution structure of CCL21 and identification of a putative CCR7 binding site

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third β-strand of CCL21's chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel antimetastatic agents.     

Plasticity comparisons between plants and animals: Concepts and mechanisms

This review attempts to present an integrated update of the issue of comparisons of phenotypic plasticity between plants and animals by presenting the problem and its integrated solutions via a whole-organism perspective within an evolutionary framework. Plants and animals differ in two important aspects: mobility and longevity. These features can have important implications for plasticity, and plasticity may even have facilitated greater longevity in plants. Furthermore, somatic genetic mosaicism, intra-organismal selection, and genomic instability contribute to the maintenance of an adaptive phenotype that is especially relevant to long-lived plants. It is contended that a cross-kingdom phylogenetic examination of sensors, messengers and responses that constitute the plasticity repertoire would be more useful than dichotomizing the plant and animal kingdoms. Furthermore, physicochemical factors must be viewed cohesively in the signal reception and transduction pathways leading to plastic responses. Comparison of unitary versus modular organisms could also provide useful insights into the range of expected plastic responses. An integrated approach that combines evolutionary theory and evolutionary history with signal-response mechanisms will yield the most insights into phenotypic plasticity in all its forms.Key words: electrical signaling, genomic instability, mechanotransduction, phenotypic plasticity, plant volatiles, reactive oxygen species, ROS signaling     

A novel role for hSMG-1 in stress granule formation

hSMG-1 is a member of the phosphoinositide 3 kinase-like kinase (PIKK) family with established roles in nonsense-mediated decay (NMD) of mRNA containing premature termination codons and in genotoxic stress responses to DNA damage. We report here a novel role for hSMG-1 in cytoplasmic stress granule (SG) formation. Exposure of cells to stress causing agents led to the localization of hSMG-1 to SG, identified by colocalization with TIA-1, G3BP1, and eIF4G. hSMG-1 small interfering RNA and the PIKK inhibitor wortmannin prevented formation of a subset of SG, while specific inhibitors of ATM, DNA-PK(cs), or mTOR had no effect. Exposure of cells to H(2)O(2) and sodium arsenite induced (S/T)Q phosphorylation of proteins. While Upf2 and Upf1, an essential substrate for hSMG-1 in NMD, were present in SG, NMD-specific Upf1 phosphorylation was not detected in SG, indicating hSMG-1's role in SG is separate from classical NMD. Thus, SG formation appears more complex than originally envisaged and hSMG-1 plays a central role in this process.     

SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate α-synuclein and are susceptible to oxidative stress

Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of α-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain.     

Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.     

Molecular Evolution of VEF-Domain-Containing PcG Genes in Plants

Arabidopsis VERNALIZATION2 （VRN2）, EMBRYONIC FLOWER2 （EMF2）, and FERTILIZATION-INDEPENDENT SEED2 （FIS2） are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes （VEF genes）, we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-1ike sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-1ike sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes, Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EM F2NRN2 divergence in accordance with species relationship. Existence of EMF2-1ike sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate seque     

Impact of Chisan (ADAPT-232) on the quality-of-life and its efficacy as an adjuvant in the treatment of acute non-specific pneumonia

A double-blind, placebo-controlled, randomized (simple randomisation), pilot (phase III) study of Chisan^® (ADAPT-232; a standardised fixed combination of extracts of Rhodiola rosea L., Schisandra chinensis Turcz. Baill., and Eleutherococcus senticosus Maxim) was carried out on two parallel groups of patients suffering from acute non-specific pneumonia. Sixty patients (males and females; 18–65 years old) received a standard treatment with cephazoline, bromhexine, and theophylline: in addition, one group of 30 patients was given Chisan mixture, whilst the second group of 30 patients received a placebo, each medication being taken twice daily from the beginning of the study for 10–15 days. The primary outcome measurements were the duration of antibiotic therapy associated with the clinical manifestations of the acute phase of the disease, together with an evaluation of mental performance in a psychometric test and the self-evaluation of quality-of-life (QOL) (WHOQOL-Bref questionnaires) before treatment and on the first and fifth days after clinical convalescence. The mean duration of treatment with antibiotics required to bring about recovery from the acute phase of the disease was 2 days shorter in patients treated with Chisan compared with those in the placebo group. With respect to all QOL domains (physical, psychological, social and ecological), patients in the Chisan group scored higher at the beginning of the rehabilitation period, and significantly higher on the fifth day after clinical convalescence, than patients in the control group. Clearly, adjuvant therapy with ADAPT-232 has a positive effect on the recovery of patients by decreasing the duration of the acute phase of the illness, by increasing mental performance of patients in the rehabilitation period, and by improving their QOL. Both the clinical and laboratory results of the present study suggest that Chisan (ADAPT-232) can be recommended in the standard treatment of patients with acute non-specific pneumonia as an adjuvant to increase the QOL and to expedite the recovery of patients.     

Dynamin 2 regulates granule exocytosis during NK cell-mediated cytotoxicity

NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.     

The initiating proteases of the complement system: controlling the cleavage

The complement system is a vital component of the host immune system, but when dysregulated, can also cause disease. The system is activated by three pathways: classical, lectin and alternative. The initiating proteases of the classical and lectin pathways have similar domain structure and employ similar mechanisms of activation. The C1r, C1s and MASP-2 proteases have the most defined roles in the activation of the system. This review focuses on the mechanisms whereby their interaction with substrates and inhibitors is regulated.