Biomedical subjects
Publications and source records attributed to P Kefalas.
The paper discusses the modelling aspects of systems with dynamic processes and dynamic structure. A combination of models bringing together the benefits of two paradigms, Population P Systems and Communicating X-machines, is introduced. A simple case study is used in order to illustrate the potential of the combined use of the two methods.
A new alkaloid derivative of [2]benzopyrano-[3,4-c] indole, isomer of 3-epimacronine, 4-methoxy-5-methyl-1,2,3,5,6,6alphaR-hexahydro-[1,3]dioxolo[4',5':6,7]isochromeno [3,4-c]indol-8-one, has been isolated from Narcissus serotinus L. (Amaryllidaceae) and its structure was elucidated by mass and spectral analysis.
The importance of honey has been recently upgraded because of its nutrient and therapeutic effect. The adulteration of honey increased exponentially in terms of both geographic and/or botanical origin. Therefore, the need has arisen for more effective quality control methods aiming at detecting adulteration. Various novel, fast, and accurate methods like AAS, HPLC, GC-MS, ES-MS, TLC, HPAED-PAD, NMR, FT-Raman, and NIR have enriched the arsenal of analytical chemist in this direction. However, apart from these novel methods, the application of multivariate analysis and, in particular, PCA, CLA, and CA, proved to be extremely useful for grouping and detecting honey of various origins. Mineral and trace element analysis were repeatedly shown to be a very effective means for the classification purposes of honey of various origins (geographical and botanical).
The aim of this paper is to prove the suitability of a parallel distributed computational model, communicating X-machines, to simulate in a natural way a well established model of molecular computation, P systems, and to present some further benefits of the approach allowing us to check for some formal properties. A set of rules to transform any P system with symbol-objects into a communicating X-machine model is presented and a variation of temporal logic for X-machines is briefly discussed, which facilitates model checking of desired properties of the system. Finally, the benefits resulting from the transformation are discussed.
Aged red wines possess significantly different polyphenolic composition compared with young ones, mainly due not only to formation of polymeric compounds but also because of oxidation, hydrolysis, and other transformations that may occur in native grape phenolics during aging. Representative Greek, single-variety, aged red wines were examined for total phenol, total flavanol, and total anthocyanin content using spectrophotometry, and attempts were made to establish correlations with the antiradical, reducing, and hydroxyl free radical scavenging activity. In addition, HPLC analyses were carried out, to ascertain whether individual polyphenols are actually responsible for the antioxidant effects of aged red wines. It was found that total flavanols are the class of polyphenols that account for hydroxyl free radical scavenging efficacy and to a lesser extent for antiradical and reducing ability, whereas there was a less significant link between the antioxidant properties and the total phenolics and only a weak relationship to total anthocyanin content. The correlation of the antioxidant properties with the principal polyphenols showed that individual compounds are weakly associated with all the antioxidant parameters, suggesting that the expression of antioxidant activity in aged red wines is rather a consequence of synergism between various phenolics, and it is not simply attributed to specific constituents.
mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane.
Full length cDNA encoding ADP-ribosyltransferase-1 (ART1) was generated from human skeletal muscle. A single base variation from the published sequence was observed (C770-->T), and was established as a polymorphism by the screening of a population of 50 Caucasians. The base variation predicted a nonconservative substitution of Leu for Pro at codon 257. Cell lines with stable and doxycycline-inducible expression of the two polymorphic forms of ART1 were generated from Chinese hamster V79 cells, and exploited in studies to compare the activities of ART1-Pro257 and ART1-Leu257. The results revealed no differences in the capacity of phosphoinositide-specific phospholipase C to cleave the two ART1 isoforms from the plasma membrane. Furthermore, the capacities of ART1-Pro257 and ART1-Leu257 to ADP-ribosylate agmatine or fibroblast growth factor-2 were similar. Differences in the catalytic activities of ART1-Pro257 and ART1-Leu257 were however, identified when measurements were made of their capacities to ADP-ribosylate membrane-associated proteins on the surface of V79 cells. Protein(s) of molecular mass 80-110 kDa were more extensively ADP-ribosylated by ART1-Pro257 than ART1-Leu257, in accordance with the Vmax (59.5 +/- 5.5 and 37.0 +/- 3.0) and Km values (12.5 +/- 4.5 and 5.0 +/- 1. 9) for ART1-Pro257 and ART1-Leu257, respectively.
The posttranslational modification of proteins by the addition of an ADP-ribose group is mediated by ADP-ribosyltransferases, which are expressed widely in many eukaryotic tissues, including leukocytes. DNA encoding arginine-specific ADP-ribosyltransferases has been cloned from both polymorphonuclear neutrophil leukocytes and lymphocytes, and their primary structures are widely conserved, particularly in those domains of the enzyme implicated in NAD+ binding and catalysis. In most cases the enzymes are tethered to the outer aspect of the cell surface or are released directly from the cell surface. The protein substrates of some of the ADP-ribosyltransferases have been identified and the catalytic activity of these enzymes has been implicated in several immune responses as well as white cell chemotaxis. This review describes recent significant advances in this field, and it seems likely that additional leukocyte functions, most particularly those linked to the activity of surface integrins, will be assigned to this class of enzymes.
1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.
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Mono(ADP-ribosyl)transferase activity has been detected on the external surface of human polymorphonuclear neutrophil leucocytes (PMNs). The corresponding cDNA has been cloned and shown to be identical to that derived from human skeletal muscle. Our results suggest that mono(ADP-ribosyl)transferase is involved in the transduction pathway mediating (i) receptor-dependent re-alignment of cytoskeletal actin and (ii) chemotaxis of PMNs.
An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1 30.4 microM and the Vmax 1.4 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP.
1. Mono(ADP-ribosyl)transferase activity has been identified on the external surface of human polymorphonuclear neutrophil leucocytes (PMNs). The enzyme is released from the plasma membrane by phosphoinositide-specific phospholipase C, suggesting a glycosylphosphatidylinositol (GPI) linkage of the enzyme to the plasma membrane. Partial sequence of cDNA encoding the enzyme suggests that it is identical to the GPI-linked mono(ADP-ribosyl)-transferase identified previously on human skeletal muscle. 2. A panel of inhibitors of mono(ADP-ribosyl)transferase (including vitamins K1 and K3, novobiocin and nicotinamide) showed a rank order of inhibitory potency similar to that described for other mono(ADP-ribosyl)transferases. Furthermore, the mono(ADP-ribosyl)ation of agmatine was inhibited also by diethylamino (benzylidineamino)guanidine (DEA-BAG), another substrate of the enzyme related structurally to arginine. 3. There was a close linear correlation between the IC50 values for inhibition of mono(ADP-ribosyl)ation of agmatine by DEA-BAG or the enzyme inhibitors and their IC50 values for inhibition of receptor-dependent polymerization of cytoskeletal actin and chemotaxis. 4. These results suggest a role for mono(ADP-ribosyl)transferase in the transduction pathway involved in receptor-dependent re-alignment of the cytoskeleton during neutrophil chemotaxis.
Expression of the c-fgr proto-oncogene is activated during differentiation of myeloid cells. We used a luciferase reporter assay to identify sequences that regulate c-fgr gene transcription during differentiation of human U937 promonocytic cells, induced by phorbol 12-myristate 13-acetate (PMA) or by tumour necrosis factor-alpha (TNF-alpha) and 1,25-dihydroxycholecalciferol (1,25-DHCC). We found that the region from nucleotides -344 to -116, with respect to the transcriptional start site, is required for basal activity of the c-fgr promoter in U937 cells, and that the region from nucleotides -1211 to -752 is responsive to PMA. No sequence elements responsive to TNF-alpha and 1,25-DHCC were found, suggesting that these agents induce c-fgr mRNA accumulation by a mechanism differing from that mediating the effects of PMA.
The c-src gene family has nine known members (blk, c-fgr, fyn, hck, lck, lyn, c-src, c-yes and yrk), each encoding a cytoplasmic protein-tyrosine kinase (PTK) believed to be involved in signal transduction. The c-src PTKs contain three domains (SH1, SH2 and SH3) that are found in many other signalling proteins. The SH1 domain has PTK activity, whilst the SH2 and SH3 domains are involved in mediating protein-protein interactions by binding to phosphotyrosine-containing and proline-rich motifs, respectively. The expression patterns of the c-src PTKs suggest that they function in a broad range of biological situations, in many cases regulating the behaviour of terminally-differentiated, post-mitotic cell types. Targeted disruption of members of the c-src family in transgenic mice has confirmed important roles for p56lck and p59fym(T) in T-lymphocyte maturation and activation, but has also revealed unexpected roles for p60c-src in bone maintenance and for p59fym(B) in learning and memory. There is increasingly detailed information about the biochemical nature of the signalling pathways in which the c-src PTKs operate and about the other signalling proteins with which they interact. The c-src PTKs can associate with activated receptor PTKs, including the receptors for platelet-derived growth factor and epidermal growth factor, by means of SH2-phosphotyrosine binding. The c-src PTKs also associated with transmembrane proteins that lack PTK activity, frequently by means of interactions involving their unique amino-terminal sequences.
Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer.