Human Protein Cluster Analysis Using Amino Acid Frequencies

The paper focuses on the development of a software tool for protein clustering according to their amino acid content. All known human proteins were clustered according to the relative frequencies of their amino acids starting from the UniProtKB/Swiss-Prot reference database and making use of hierarchical cluster analysis. RESULTS were compared to those based on sequence similarities.Proteins display different clustering patterns according to type. Many extracellular proteins with highly specific and repetitive sequences (keratins, collagens etc.) cluster clearly confirming the accuracy of the clustering method. In our case clustering by sequence and amino acid content overlaps. Proteins with a more complex structure with multiple domains (catalytic, extracellular, transmembrane etc.), even if classified very similar according to sequence similarity and function (aquaporins, cadherins, steroid 5-alpha reductase etc.) showed different clustering according to amino acid content. Availability of essential amino acids according to local conditions (starvation, low or high oxygen, cell cycle phase etc.) may be a limiting factor in protein synthesis, whatever the mRNA level. This type of protein clustering may therefore prove a valuable tool in identifying so far unknown metabolic connections and constraints

Production of platelet-activating factor by chick retina.

In the present study it is demonstrated that platelet-activating factor (PAF) was produced by chick retinas, upon stimulation with neurotransmitters such as acetylcholine (ACh), dopamine, or with calcium ionophore A23187, but not upon stimulation with gamma-amino-n-butyric acid, L-glycine, L-glutamate, epinephrine, or histamine. PAF produced in response to ACh, dopamine, or A23187 was not released into supernatants but was extractable from retinas. The amounts of extractable PAF increased after sonication of stimulated retinas. While no PAF activity could be recovered from unstimulated retinas, small amounts of this lipid can be detected following sonication of the tissue. The amount of extractable PAF from ACh-, dopamine-, or A23187-stimulated retinas was dependent upon the incubation time and concentration of the agonists. PAF was identified on the basis of chemical and lipase treatments, biological activity with washed rabbit platelets, behavior on thin layer chromatography, and high pressure liquid chromatography. Control cell preparations (leukocytes, erythrocytes, and embryogenic fibroblasts) did not produce PAF upon neurotransmitter stimulation. ACh and dopamine promoted PAF production by increasing dithiothreitol-insensitive cholinephosphotransferase activity, without affecting the acetyltransferase activity. In contrast, the A23187 ionophore stimulated the acetyltransferase activity but did not affect the dithiothreitol-insensitive cholinephosphotransferase

Mitochondrial Localization of Vitamin D Receptor in Human Platelets and Differentiated Megakaryocytes

Background: Like other steroid hormones, vitamin D elicits both transcriptional events and rapid non genomic effects. Vitamin D receptor (VDR) localization and mechanisms of VDR-triggered non genomic responses are still controversial. Although anticoagulant effects of vitamin D have been reported and VDR signalling has been characterized in monocytes and vascular cells, nothing is known about VDR expression and functions in human platelets, anucleated fragments of megakaryocytes which are known targets of other steroids. Methodology/Principal Findings: In this study we characterized the expression and cellular localization of VDR in human platelets and in a megakaryocyte lineage. Human platelets and their TPA-differentiated precursors expressed a classical 50 kDa VDR protein, which increased with megakaryocytes maturation. By biochemical fractionation studies we demonstrated the presence of the receptor in the soluble and mitochondrial compartment of human platelets, and the observation was confirmed by immunoelectron microscopy analysis. Similar localization was found in mature megakaryocytes, where besides its classical nuclear localization the receptor was evident as soluble and mitochondria resident protein. Conclusions: The results reported here suggest that megakaryocytopoiesis and platelet activation, which are calciumdependent events, might be modulated by a mitochondrial non genomic activity of VDR. These data open challengin

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells.

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells

Stimulation of the Na+/H+ exchanger in human endothelial cells activated by granulocyte- and granulocyte-macrophage-colony-stimulating factor. Evidence for a role in proliferation and migration.

It has been shown that human endothelial cells (HEC) are stimulated to migrate and proliferate by granulocyte (G)- and granulocyte-macrophage (GM)-colony-stimulating factor (CSF) (Bussolino, F., Wang, J. M., Defilipii, P. Turrini, F., Sanavio, F., Edgell, C.-J. S., Aglietta, M., Arese, P., and Mantovani, A. (1989) Nature 337, 471-473). The rapid intracellular events initiated by these cytokines on binding to their receptors on HEC are not defined. Addition of G- or GM-CSF to HEC produced a rapid activation of Na+/H+ exchanger resulting in an increase in intracellular pH (pHi). Both cytokines induced an alkaline displacement in the pHi dependence of the exchanger without affecting the affinity for external Na+ (Nao) and the rate of exchanger. Ethylisopropylamiloride, a selective inhibitor of the Na+/H+ exchanger, inhibited the intracellular alkalinization, the migration, and proliferation induced by G- and GM-CSF. The data indicate that G- and GM-CSF initiate a rapid exchange of Na+ and H+ by means of the Na+/H+ exchanger and that this ethylisopropylamiloride-sensitive ions flux is important to the biological effects of these cytokines on HEC

Targeted knowledge: interaction and rich user experience towards a scholarly communication that "lets"

All living systems share many properties including hardly predictable behaviours, due to the differences between individuals and the chaos in natural environments. The reductionist approach to the interpretation of these phenomena suffers from the oversimplification of the factors involved in the quest of universal “scientific” explanations. The validation of scientific paradigms is based on the consensus of leading groups that decide what is true and what is not. That means that all events - not only conflicting opinions but also conflicting raw data - not fitting with the scientific official truth were never published, and that supported indirectly the correctness of the experts’ choice. With the advent of Web 2.0 and the freedom of publishing, the number of these not fitting events has dramatically increased. Yesterday, data were supplied to the reader with the interpretation. Now the reader has to afford in each field a huge amount of data and opinions. Extracting from the garbage the information you need requires a strategy. Strategy is a science by itself. In the specific case of knowledge the first step is to define knowledge. The aim of life sciences, medicine, social sciences is to modify the reality when it is no longer sustainable, whatever it could mean in every single situation. I have to know how my system works to modify it. Knowledge is the information that allows me to succeed in my tasks. Tasks must have an assessable target. All information useful and therefore processed to attain the target will be “targeted knowledge”. Information can be selected on the basis of their congruence with the rules internal to the system. In Web 2.0 we found proper tools to test this approach. We implemented a web application - whose aim is an easier identification of the molecular basis of the diseases - structured in Rules, Reports, Items, Pathways and Tools referring and linking one another. The use of tags allows and fosters a free and personal use of information to create original knowledge. Users can follow and open innovative paths each time answering a different question, re-combining the fitting information. Our application is an example of advanced Problem Solving: the patient as a whole, not as a single symptom, has to be understood as a part of the living world (Gaia, with its rules) whose components (Items, Pathways) are described in their multiple roles and connections. The Web allows easy access to information, the program allows the network creation, the Rules drive the selection of the information and become more and more stable the more they evolutionary adapt to the reality. Something like the DNA, carrying sequences millions years old in an ever changing world