Genome‐wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice

We conducted genome‐wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3‐binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3‐associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin.     

Dimethylaminopyridine derivatives of lupane triterpenoids are potent disruptors of mitochondrial structure and function

Development of mitochondrially-targeted drugs is receiving increasing attention because of the central roles these organelles play in energy production, reactive oxygen generation, and regulation of cell death pathways. Previous studies have demonstrated that both natural and synthetic triterpenoids can disrupt mitochondrial structure and function. In this study, we tested the ability of a number of dimethylaminopyridine (DMAP) derivatives of lupane triterpenoids to target mitochochondria in two human melanoma cell lines and an untransformed normal fibroblast line. These compounds induced a striking fragmentation and depolarization of the mitochondrial network, along with an inhibition of cell proliferation. A range of potencies among these compounds was noted, which was correlated with the number, position, and orientation of the DMAP groups. Overall, the extent of proliferation inhibition mirrored the effectiveness of mitochondrial disruption. Thus, DMAP derivatives of lupane triterpenoids can be potent mitochondrial perturbants that appear to suppress cell growth primarily via their mitochondrial effects.     

The impact of Ivan Málek’s continuous culture concept on bioprocessing

This paper summarizes research results and their industrial applications obtained by continuous culture in the former Czechoslovakia. Past achievements as well as recent trends and developments worldwide are presented. The term “Prague School of continuous culture” is put forward and its international activity is outlined. The impact of this school was pervasive across the entire field of applied microbiology and biotechnology in Czechoslovakia and, perhaps, even beyond the country's boarders. Continuous culture is a very mature field, and since its establishment it has become a powerful research tool. The present activity in this field amounts to a renaissance of continuous culture, emphasizing new dimensions in bioinformatics and systems biology.     

Interactions between glycolytic enzymes of Mycoplasma pneumoniae

With only 688 protein-coding genes, Mycoplasma pneumoniae is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in M. pneumoniae and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.     

Exposure of beta-tubulin regions defined by antibodies on an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>microtubule protofilament model and in the cells

Background  

The function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited.     

Proteome changes in platelets activated by arachidonic acid,collagen, and thrombin

Background  

Platelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS.     

Differential expression and cellular distribution of γ‐tubulin and βIII‐tubulin in medulloblastomas and human medulloblastoma cell lines

The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neurological disorders such as cerebral ischemia and Alzheimer's disease. Many in vitro BBB models derived from animal tissues have been established to elucidate the mechanism of BBB insufficiency. However, only a few human immortalized in vitro BBB models have been reported. In the present study, a temperature‐sensitive SV40‐T antigen was introduced to immortalize cells using a retrovirus to obtain a better human in vitro BBB model which sustains physiological properties. This endothelial cell (EC) line, termed TY08, showed a spindle‐shaped morphology. The cells expressed all key tight junctional proteins, such as occludin, claudin‐5, zonula occludens (ZO)‐1 and ZO‐2 at their cell‐to‐cell boundaries, and had low permeability to inulin across its monolayer. The cells also expressed various influx and efflux transporters and exhibited the functional expression of p‐glycoprotein. Furthermore, the TY08 cells grew and proliferated well under the permissive temperature and stopped growing under the non‐permissive temperature to serve as physiological ECs forming the BBB. Thus, conditionally immortalized TY08 cells retaining the in vivo BBB functions should facilitate analyses for determining the pathophysiology of various neurological diseases. J. Cell. Physiol. 225: 519–528, 2010. © 2010 Wiley‐Liss, Inc.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.