Types of potentials in a mitotic spindle

The dynamics of a mitotic spindle is very important to understand if the functioning of mitosis has to be understood and defined very accurately. There are a number of forces involved in such a process. Despite of the fact that there have been numerous studies done on the functioning of a mitotic spindle, there is still not a very precise understanding of this system and how it behaves. This study aims at understanding and expressing all the possible potentials which might be responsible in a mitotic spindle and its mechanism.     

Arp2/3 promotes junction formation and maintenance in the Caenorhabditis elegans intestine by regulating membrane association of apical proteins

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin-radixin-moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes.     

Spreading depression sends microglia on Lévy flights

Spreading depression (SD) is thought to cause migraine aura, and perhaps migraine, and includes a transient loss of synaptic activity preceded and followed by increased neuronal excitability. Activated microglia influence neuronal activity and play an important role in homeostatic synaptic scaling via release of cytokines. Furthermore, enhanced neuronal function activates microglia to not only secrete cytokines but also to increase the motility of their branches, with somata remaining stationary. While SD also increases the release of cytokines from microglia, the effects on microglial movement from its synaptic activity fluctuations are unknown. Accordingly, we used time-lapse imaging of rat hippocampal slice cultures to probe for microglial movement associated with SD. We observed that in uninjured brain whole microglial cells moved. The movements were well described by the type of Lévy flight known to be associated with an optimal search pattern. Hours after SD, when synaptic activity rose, microglial cell movement was significantly increased. To test how synaptic activity influenced microglial movement, we enhanced neuronal activity with chemical long-term potentiation or LPS and abolished it with TTX. We found that microglial movement was significantly decreased by enhanced neuronal activity and significantly increased by activity blockade. Finally, application of glutamate and ATP to mimic restoration of synaptic activity in the presence of TTX stopped microglial movement that was otherwise seen with TTX. Thus, synaptic activity retains microglial cells in place and an absence of synaptic activity sends them off to influence wider expanses of brain. Perhaps increased microglial movements after SD are a long-lasting, and thus maladaptive, response in which these cells increase neuronal activity via contact or paracrine signaling, which results in increased susceptibility of larger brain areas to SD. If true, then targeting mechanisms that retard activity-dependent microglial Lévy flights may be a novel means to reduce susceptibility to migraine.     

Leptin Regulates KATP Channel Trafficking in Pancreatic β-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)

Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (K_ATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing K_ATP channel conductance in β-cells. We investigated the mechanism by which leptin increases K_ATP channel conductance. We show that leptin causes a transient increase in surface expression of K_ATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on K_ATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on K_ATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates K_ATP channel trafficking to modulate β-cell function and insulin secretion.     

Distamycin-stabilized Antiparallel-Parallel Combination (APC) DNA

Abstract

The formation of Antiparallel-Parallel-Combination (APC) DNA, a liner duplex with a segment of parallel-stranded (ps) helix flanked by conventional B-DNA, was tested with a number of synthetic oligonucleotides. The groove-binding ligand distamycin A (DstA) was used to stabilize the ps segment comprising five A·T base pairs. Two drug molecules bound per APC, one in each of the two equivalent grooves characteristic of ps-DNA. APC-DNA, reference molecules and their complexes with DstA were analysed by several methods: circular dichroism and absorption spectroscopy, thermal denaturation, chemical modification, and molecular modeling. The dye binding stoichiometry differed significantly due to inherent structural differences in the groove geometries of ps-DNA (trans base pairs, similar grooves) and conventional antiparallel-stranded (aps) B-DNA (cis base pairs, distinct major and minor grooves). The data support the existence of APC folding in solution.     

The Structure of Microbial Community and Degradation of Diatoms in the Deep Near-Bottom Layer of Lake Baikal

Insight into the role of bacteria in degradation of diatoms is important for understanding the factors and components of silica turnover in aquatic ecosystems. Using microscopic methods, it has been shown that the degree of diatom preservation and the numbers of diatom-associated bacteria in the surface layer of bottom sediments decrease with depth; in the near-bottom water layer, the majority of bacteria are associated with diatom cells, being located either on the cell surface or within the cell. The structure of microbial community in the near-bottom water layer has been characterized by pyrosequencing of the 16S rRNA gene, which has revealed 149 208 unique sequences. According to the results of metagenomic analysis, the community is dominated by representatives of Proteobacteria (41.9%), Actinobacteria (16%); then follow Acidobacteria (6.9%), Cyanobacteria (5%), Bacteroidetes (4.7%), Firmicutes (2.8%), Nitrospira (1.6%), and Verrucomicrobia (1%); other phylotypes account for less than 1% each. For 18.7% of the sequences, taxonomic identification has been possible only to the Bacteria domain level. Many bacteria identified to the genus level have close relatives occurring in other aquatic ecosystems and soils. The metagenome of the bacterial community from the near-bottom water layer also contains 16S rRNA gene sequences found in previously isolated bacterial strains possessing hydrolytic enzyme activity. These data show that potential degraders of diatoms occur among the vast variety of microorganisms in the near-bottom water of Lake Baikal.     

ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages

Differentiation to different types of macrophages determines their distinct functions. Tumor-associated macrophages (TAMs) promote tumorigenesis owing to their proangiogenic and immune-suppressive functions similar to those of alternatively activated (M2) macrophages. We report that reactive oxygen species (ROS) production is critical for macrophage differentiation and that inhibition of superoxide (O²⁻) production specifically blocks the differentiation of M2 macrophages. We found that when monocytes are triggered to differentiate, O²⁻ is generated and is needed for the biphasic ERK activation, which is critical for macrophage differentiation. We demonstrated that ROS elimination by butylated hydroxyanisole (BHA) and other ROS inhibitors blocks macrophage differentiation. However, the inhibitory effect of ROS elimination on macrophage differentiation is overcome when cells are polarized to classically activated (M1), but not M2, macrophages. More importantly, the continuous administration of the ROS inhibitor BHA efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in mouse cancer models. Targeting TAMs by blocking ROS can be a potentially effective method for cancer treatment.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi

The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesise that they might be involved in the Wolbachia-Brugia symbiotic relationship. Notably, immunolocalisation studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localisation by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.