Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Csk homologous kinase (CHK), unlike Csk, enhances MAPK activation via Ras-mediated signaling in a Src-independent manner

Substantial evidence exists supporting the notion that Csk and CHK, two negative regulatory kinases of the Src tyrosine kinase family, play distinct roles during development of the nervous system. One of the differences relies on the effects of both kinases on the MAPK transduction pathway. Specifically, CHK was shown to enhance MAPK signaling, while the role of Csk was unclear. In this work, we compared the effect of CHK versus Csk on MAPK signaling and elucidated the signaling pathway mediated by CHK leading to the activation of Erk1/2. Exogenous expression of wild-type CHK, but not Csk or a dead-kinase mutant of CHK, resulted in enhanced Erk1/2 phosphorylation in PC12 cells. CHK inhibited Src activity following stimulation of the cells with NGF. However, stimulation of Erk1/2 activation by CHK was independent of the NGF stimulation or the inhibition of Src kinase by CHK. CHK induced a complex formation between SHP-2 and Grb2, subsequently leading to the increased activity of Ras as well as Erk1/2 activation via the Raf/MEK1/2 pathway. Down-regulation of the expression of endogenous CHK by RNAi in PC12 cells led to a significant decrease in MAPK activation following NGF stimulation. Stimulation of CHK-overexpressing PC12 cells with EGF induced neurite outgrowth in the majority of cells. Taken together, this study describes for the first time the Src-independent actions of CHK and provides novel insights into CHK function in neural cells.     

FUSION OF INTACT HUMAN ERYTHROCYTES AND ERYTHROCYTE GHOSTS

Sendai virus is able to induce the fusion of human erythrocytes. Bivalent cations or ATP are not essential for polyerythrocyte formation. High fusion indices were obtained when Sendai virus was added to cells incubated in the presence of both EDTA and iodoacetic acid. Human erythrocyte ghosts prepared by gradual hemolysis still retain the potential to undergo virus-induced fusion. Fusion of human red blood cells without the addition of viruses was obtained by incubation of erythrocytes at pH 10.5 in the presence of Ca⁺⁺ (40 mM) or by addition of phospholipase C Clostridium perfringens preparations to cells previously agglutinated or polylysine.     

An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non‐human primates, demonstrating the strain''s ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.     

Mechanisms and Fitness Costs of Resistance to Antimicrobial Peptides LL-37, CNY100HL and Wheat Germ Histones

Antimicrobial peptides (AMPs) represent a potential new class of antimicrobial drugs with potent and broad-spectrum activities. However, knowledge about the mechanisms and rates of resistance development to AMPs and the resulting effects on fitness and cross-resistance is limited. We isolated antimicrobial peptide (AMP) resistant Salmonella typhimurium LT2 mutants by serially passaging several independent bacterial lineages in progressively increasing concentrations of LL-37, CNY100HL and Wheat Germ Histones. Significant AMP resistance developed in 15/18 independent bacterial lineages. Resistance mutations were identified by whole genome sequencing in two-component signal transduction systems (pmrB and phoP) as well as in the LPS core biosynthesis pathway (waaY, also designated rfaY). In most cases, resistance was associated with a reduced fitness, observed as a decreased growth rate, which was dependent on growth conditions and mutation type. Importantly, mutations in waaY decreased bacterial susceptibility to all tested AMPs and the mutant outcompeted the wild type parental strain at AMP concentrations below the MIC for the wild type. Our data suggests that resistance to antimicrobial peptides can develop rapidly through mechanisms that confer cross-resistance to several AMPs. Importantly, AMP-resistant mutants can have a competitive advantage over the wild type strain at AMP concentrations similar to those found near human epithelial cells. These results suggest that resistant mutants could both be selected de novo and maintained by exposure to our own natural repertoire of defence molecules.     

A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II

Primary sequences of proteins often contain motifs that serve as "signatures" for subcellular targeting, such as a nuclear localization signal (NLS). However, many nuclear proteins do not harbor a recognizable NLS, and the pathways that mediate their nuclear translocation are unknown. This work focuses on CRABP-II, a cytosolic protein that moves to the nucleus upon binding of retinoic acid. While CRABP-II does not contain an NLS in its primary sequence, such a motif could be recognized in the protein's tertiary structure. We map the retinoic acid-induced structural rearrangements that result in the presence of this NLS in holo- but not apo-CRABP-II. The signal, whose three-dimensional configuration aligns strikingly well with a "classical" NLS, mediates ligand-induced association of CRABP-II with importin alpha and is critical for nuclear localization of the protein. The ligand-controlled NLS "switch" of CRABP-II may represent a general mechanism for posttranslational regulation of the subcellular distribution of a protein.     

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.     

An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies

Background  

We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol.     

DNA polymerase of mitochondria is a gamma-polymerase.

Mitochondria isolated from rat liver cells or mycoplasma-free HeLa cells contain a single DNA polymerase activity which is closely related to, or identical to, the DNA polymerase gamma activity found in the homologous cell. In rat liver cells, about 16% of the total cytoplasmic gamma-polymerase activity is found associated with mitochondria and in HeLa cells about 20% of the total cellular gamma-polymerase is mitochondria associated. Since mitochondria possess no unique DNA polymerase activity, the number of DNA polymerases now known in mammalian cells is reduced, from the previously proposed four enzymes, to three--DNA polymerases alpha, beta, and gamma.     

Data Mining of Molecular Dynamics Trajectories of Nucleic Acids

Abstract

Analysis, storage, and transfer of molecular dynamic trajectories are becoming the bottleneck of computer simulations. In this paper we discuss different approaches for data mining and data processing of huge trajectory files generated from molecular dynamic simulations of nucleic acids.