Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

Genetic Deletion of ACE2 Induces Vascular Dysfunction in C57BL/6 Mice: Role of Nitric Oxide Imbalance and Oxidative Stress

Accumulating evidence indicates that angiotensin-converting enzyme 2 (ACE2) plays a critical role in cardiovascular homeostasis, and its altered expression is associated with major cardiac and vascular disorders. The aim of this study was to evaluate the regulation of vascular function and assess the vascular redox balance in ACE2-deficient (ACE2^-/y) animals. Experiments were performed in 20–22 week-old C57BL/6 and ACE2^-/y male mice. Evaluation of endothelium-dependent and -independent relaxation revealed an impairment of in vitro and in vivo vascular function in ACE2^-/y mice. Drastic reduction in eNOS expression at both protein and mRNA levels, and a decrease in ^•NO concentrations were observed in aortas of ACE2^-/y mice in comparison to controls. Consistently, these mice presented a lower plasma and urine nitrite concentration, confirming reduced ^•NO availability in ACE2-deficient animals. Lipid peroxidation was significantly increased and superoxide dismutase activity was decreased in aorta homogenates of ACE2^-/y mice, indicating impaired antioxidant capacity. Taken together, our data indicate, that ACE2 regulates vascular function by modulating nitric oxide release and oxidative stress. In conclusion, we elucidate mechanisms by which ACE2 is involved in the maintenance of vascular homeostasis. Furthermore, these findings provide insights into the role of the renin-angiotensin system in both vascular and systemic redox balance.     

A hominid tooth from Bulgaria: the last pre-human hominid of continental Europe

A hominid upper premolar was discovered in the Azmaka quarry, near Chirpan (Bulgaria). The associated fauna, especially the co-occurrence of Choerolophodon and Anancus among the proboscideans, and Cremohipparion matthewi and Hippotherium brachypus among the hipparions, constrains the age of the locality to the second half of the middle Turolian (ca. 7 Ma), making it the latest pre-human hominid of continental Europe and Asia Minor. The available morphological and metric data are more similar to those of Ouranopithecus from the Vallesian of Greece than to those of the early to middle Turolian hominids of Turkey and Georgia, but the time gap speaks against a direct phyletic link, and Turolian migration from the east cannot be rejected.     

Differences in endocytosis and intracellular sorting of ricin and viscumin in 3T3 cells

Ricin and viscumin are heterodimeric protein toxins. Their A-chain is enzymatically active and removes an adenine residue from the 28S rRNA, the B-chain has lectin activity and binds to terminal galactose residues of cell surface receptors. The toxins reveal a high degree of identity in their amino acid sequences. Nevertheless, uptake into 3T3 cells occurs via different receptors and endocytotic pathways. This has been revealed by enzyme linked based analysis of ricin competition with viscumin, and by fluorochrome-labeled toxins (viscumin-FITC, ricin-Alexa 568), which were added simultaneously or separately to living cells. Then the uptake was followed by confocal laser scanning microscopy. Ricin immediately is delivered to the tubular and vesicular structures of endosomes in the perinuclear area while viscumin becomes endocytosed into small vesicles preferentially in the cell periphery. After about 60 min both these toxins may be found in tubo-vesicular structures of endosomes where the sorting process can directly be observed. The fact that this sorting takes place is a strong argument for the assumption that the toxins are bound to membrane proteins, either to their original receptors or to other proteins inside the endosomal compartment exhibiting terminal galactose residues. The toxins are biologically fully active as has been proven by binding and by toxicity experiments, thus the differences in targeting do not arise from labeling.     

Large-scale de novo prediction of physical protein-protein association

Information about the physical association of proteins is extensively used for studying cellular processes and disease mechanisms. However, complete experimental mapping of the human interactome will remain prohibitively difficult in the near future. Here we present a map of predicted human protein interactions that distinguishes functional association from physical binding. Our network classifies more than 5 million protein pairs predicting 94,009 new interactions with high confidence. We experimentally tested a subset of these predictions using yeast two-hybrid analysis and affinity purification followed by quantitative mass spectrometry. Thus we identified 462 new protein-protein interactions and confirmed the predictive power of the network. These independent experiments address potential issues of circular reasoning and are a distinctive feature of this work. Analysis of the physical interactome unravels subnetworks mediating between different functional and physical subunits of the cell. Finally, we demonstrate the utility of the network for the analysis of molecular mechanisms of complex diseases by applying it to genome-wide association studies of neurodegenerative diseases. This analysis provides new evidence implying TOMM40 as a factor involved in Alzheimer's disease. The network provides a high-quality resource for the analysis of genomic data sets and genetic association studies in particular. Our interactome is available via the hPRINT web server at: www.print-db.org.     

Exposure of Chlorpromazine to 266 nm Laser Beam Generates New Species with Antibacterial Properties: Contributions to Development of a New Process for Drug Discovery

Introduction

Phenothiazines when exposed to white light or to UV radiation undergo a variety of reactions that result in degradation of parental compound and formation of new species. This process is slow and may be sped up with exposure to high energy light such as that produced by a laser.

Methods

Varying concentrations of Chlorpromazine Hydrochloride (CPZ) (2–20 mg/mL in distilled water) were exposed to 266 nm laser beam (time intervals: 1–24 hrs). At distinct intervals the irradiation products were evaluated by spectrophotometry between 200–1500 nm, Thin Layer Chromatography, High Pressure Liquid Chromatography (HPLC) - Diode Array Detection, HPLC tandem mass spectrometry, and for activity against the CPZ sensitive test organism Staphylococcus aureus ATCC 25923.

Results

CPZ exposure to 266 nm laser beam of given energy levels yielded species, whose number increased with duration of exposure. Although the major species produced were Promazine (PZ), hydroxypromazine or PZ sulfoxide, and CPZ sulfoxide, over 200 compounds were generated with exposure of 20 mg/mL of CPZ for 24 hrs. Evaluation of the irradiation products indicated that the bioactivity against the test organism increased despite the total disappearance of CPZ, that is due, most probably, to one or more new species that remain yet unidentified.

Conclusions

Exposure of CPZ to a high energy (6.5 mJ) 266 nm laser beam yields rapidly a large number of new and stable species. For biological grade phenothiazines (in other words knowing the impurities in the samples: solvent and solute) this process may be reproducible because one can control within reasonably low experimental errors: the concentration of the parent compound, the laser beam wavelength and average energy, as well as the duration of the exposure time. Because the process is “clean” and rapid, it may offer advantages over the pyrogenically based methods for the production of derivatives.     

Zearalenone Mycotoxin Affects Immune Mediators,MAPK Signalling Molecules,Nuclear Receptors and Genome-Wide Gene Expression in Pig Spleen

The toxicity of zearalenone (ZEA) was evaluated in swine spleen, a key organ for the innate and adaptative immune response. Weaned pigs were fed for 18 days with a control or a ZEA contaminated diet. The effect of ZEA was assessed on wide genome expression, pro- (TNF-α, IL-8, IL-6, IL-1β, IFN-γ) and anti-inflammatory (IL-10, IL-4) cytokines, other molecules involved in inflammatory processes (MMPs/TIMPs), as well as signaling molecules, (p38/JNK1/JNK2-MAPKs) and nuclear receptors (PPARγ/NFkB/AP-1/STAT3/c-JUN). Microarray analysis showed that 46% of total number of differentially expressed genes was involved in cellular signaling pathway, 13% in cytokine network and 10% in the inflammatory response. ZEA increased expression and synthesis of pro- inflammatory (TNF-α, IL-8, IL-6, IL-1β) and had no effect on IFN-γ, IL-4 and IL-10 cytokines in spleen. The inflammatory stimulation might be a consequence of JNK pathway activation rather than of p-38MAPK and NF-kB involvement whose gene and protein expression were suppressed by ZEA action. In summary, our findings indicated the role of ZEA as an immune disruptor at spleen level.     

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug – clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.     

Expression of human‐specific ARHGAP11B in mice leads to neocortex expansion and increased memory flexibility

Neocortex expansion during human evolution provides a basis for our enhanced cognitive abilities. Yet, which genes implicated in neocortex expansion are actually responsible for higher cognitive abilities is unknown. The expression of human‐specific ARHGAP11B in embryonic/foetal mouse, ferret and marmoset neocortex was previously found to promote basal progenitor proliferation, upper‐layer neuron generation and neocortex expansion during development, features commonly thought to contribute to increased cognitive abilities. However, a key question is whether this phenotype persists into adulthood and if so, whether cognitive abilities are indeed increased. Here, we generated a transgenic mouse line with physiological ARHGAP11B expression that exhibits increased neocortical size and upper‐layer neuron numbers persisting into adulthood. Adult ARHGAP11B‐transgenic mice showed altered neurobehaviour, notably increased memory flexibility and a reduced anxiety level. Our data are consistent with the notion that neocortex expansion by ARHGAP11B, a gene implicated in human evolution, underlies some of the altered neurobehavioural features observed in the transgenic mice, such as the increased memory flexibility, a neocortex‐associated trait, with implications for the increase in cognitive abilities during human evolution.