A novel fragment derived from the beta chain of human fibrinogen, beta43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo.

BACKGROUND: Angiogenesis and haemostasis are closely linked within tumours with many haemostatic proteins regulating tumour angiogenesis. Indeed we previously identified a fragment of human fibrinogen, fibrinogen E-fragment (FgnE) with potent anti-angiogenic properties in vitro and cytotoxic effects on tumour vessels in vivo. We therefore investigated which region of FgnE was mediating vessel cytotoxicity. METHODS: Human dermal microvascular endothelial cells (ECs) were used to test the efficacy of peptides derived from FgnE on proliferation, migration, differentiation, apoptosis and adhesion before testing the efficacy of an active peptide on tumour vasculature in vivo. RESULTS: We identified a 20-amino-acid peptide derived from the beta chain of FgnE, beta43-63, which had no effect on EC proliferation or migration but markedly inhibited the ability of activated ECs to form tubules or to adhere to various constituents of the extracellular matrix - collagen IV, fibronectin and vitronectin. Furthermore, our data show that beta43-63 interacts with ECs, in part, by binding to alpha(v)beta(3), so soluble alpha(v)beta(3) abrogated beta43-63 inhibition of tubule formation by activated ECs. Finally, when injected into mice bearing tumour xenografts, beta43-63 inhibited tumour vascularisation and induced formation of significant tumour necrosis. CONCLUSIONS: Taken together, these data suggest that beta43-63 is a novel anti-tumour peptide whose anti-angiogenic effects are mediated by alpha(v)beta(3).

The influence of depleted glutathione levels on the photodynamic action of zinc phthalocyanine in CHO K1 cells.

OBJECTIVE: The current study focuses on any influence that depletion of endogenous glutathione in CHO K1 cells may have on the photodynamic action of zinc phthalocyanine (ZnPc). MATERIALS AND METHODS: Two lasers--a HeNe laser, 632.5 nm, maximum power output 3.5 mW, and a Toshiba semiconducting laser, 670 nm, maximum power of 7 mW--were used. Chinese Hamster Ovary cells (CHO K1) were exposed to light, 2-10 J. Cellular reduced glutathione levels [GSH] were depressed prior to exposure to ZnPc and laser light, using buthionine sulphoximine, a potent inhibitor of gamma-glutamylcysteine synthetase. The influence of hypoxic intracellular conditions was studied by reduction of oxygen content of cells by 80% following purging of cell cultures with nitrogen. RESULTS: In well-aerated cells, doubling times are reduced by the photodynamic action of ZnPc by 29 +/- 6%, fig 2 (p = 0.01). Cells with lowered [GSH] do not show this effect (p = 0.1). When hypoxic cells are studied at normal [GSH], no photodynamic effect is observed (p = 0.1). When cell viability is studied, using the 670-nm laser, a photodynamic effect is observed, (80% fall from controls, p < 0.001), irrespective of the cellular [GSH] level for a single dose of 6 J. This effect is observed in cells with normal [GSH], for varied doses of 2 J and higher (63% fall at 2 J, p < 0.001). CONCLUSIONS: Lowered [GSH] was observed to depress the photodynamic effect of ZnPc when cell-doubling times were the endpoint. The photostimulating effect of ZnPc was similarly suppressed by hypoxic conditions. When cell viability was the endpoint, then a photodynamic effect of ZnPc was observed irrespective of the endogenous [GSH] values.

Photodynamically induced changes of acetylcholinesterase activity from human erythrocytes.

The light and photodynamic actions on acetylcholinesterase activity from human erythrocytes were studied. After light irradiation (670 nm, semiconducting low power laser) the maximum reaction rate increased from 13.3 to 14.8 mumol per ml packed cells per min for an energy dose 9 J, and above that dose it decreased (10.8 mumol per ml packed cells per min for an energy dose of 15 J). The Michaelis-Menten constant changed in the opposite direction. After irradiation of erythrocyte suspension in the presence of zinc phthalocyanine the reaction rate increased, reaching the maximum for energy dose of 0.75 J (16.85 in comparison to the control value of 14.7 mumol per ml packed cells per min. Similarly, the Michaelis-Menten constant decreased reaching a minimum for an energy dose of 0.75 J (0.04 mM compared to 0.07 mM for control). Incubation of erythrocytes with the dye in the dark increased the reaction rate from 13.3 to 14.7 mumol per ml packed cells per min. Neither the incubation in the dark nor irradiation with laser light caused changes of enzyme activity in the presence of chloroaluminium or metal-free phthalocyanine.

Effect of combined treatment with perindoprilat and low-power red light laser irradiation on human erythrocyte membrane fluidity, membrane potential and acetylcholinesterase activity.

Erythrocyte membrane fluidity, membrane potential and acetylcholinesterase activity were estimated after in vitro combined treatment of human erythrocytes with perindoprilat and low-power red light irradiation. Membrane fluidity was determined using fluorescent labels spectroscopy; membrane potential was evaluated by means of potential-sensitive fluorescent dyes; and acetylcholinesterase activity was estimated using the Ellman method. Both perindoprilat and laser irradiation, when used separately, increase microviscosity in the polar region and hyperpolarize the membranes in comparison with control erythrocytes. The combined action of these agents does not cause any further change in these parameters. Perindoprilat has an additional inhibitory effect on the activity of acetylcholinesterase, whereas laser irradiation causes an increase in the activity of the enzyme. Their combined action restores the initial activity of the enzyme independently of the sequence of treatment with both agents.

Interaction of perindoprilat with human red blood cells.

The effects of perindoprilat on the morphology and dynamic properties of human erythrocytes were studied by light microscopy, electron spin resonance spectroscopy and spectrophotometric methods. Erythrocytes were exposed to perindoprilat at 37 degrees C for 30 and 120 min. It was shown that the drug at a concentration of 0.75 microg/ml did not cause significant changes in the structure of erythrocyte membranes. Higher doses of the drug (7.5 and 75 microg/ml) induced changes in membrane fluidity in the hydrophobic core of the lipid bilayer, the conformation of membrane proteins, the number of SH groups and the activity of membrane-bound acetylcholinesterase (AChE). These modifications were accompanied by changes in the shape of erythrocytes and did not depend on time of incubation. Therefore, it is proposed that perindoprilat perturbs the lipid bilayer and disturbs the organization of the protein-lipid environment.

Acetylcholinesterase activity of normal and diabetic human erythrocyte membranes: the effect of oxidative agents.

The activity characteristics of membrane acetylcholinesterase from red blood cells of diabetic patients are very different from those of healthy donors: the limiting enzyme reaction rate is 17.2 +/- 0.8 mumol acetylthiocholine per ml packed cells per min compared with 13.1 +/- 0.8 mumol for control cells. This Michaelis constants for substrate are the same: 0.061 +/- 0.007 mM for diabetic and 0.061 +/- 0.004 mM for control cells. Cell exposure to oxidative agent (t-butyl hydroperoxide) significantly changes the enzyme activity parameters. The limiting enzyme reaction rate increases but the affinity for the substrate decreases at lower oxidant concentrations (up to 0.1 mM for the "diabetic" erythrocytes and up to 0.4 mM for the control ones). At higher oxidant concentrations both the limiting reaction rate and the Michaelis constant decrease. The susceptibility of erythrocyte membranes of diabetic patients to oxidative stress is much higher in comparison with control erythrocyte membranes.

Pyrimidine ribonucleoside phosphorylase activity vs 5- and/or 6-substituted uracil and uridine analogues, including conformational aspects.

The pyrimidine ribonucleoside phosphorylase from Salmonella typhimurium phosphorylyses 6-methyluridine, a uridine analogue sterically constrained to the syn conformation about the glycosylic bond, as effectively as uridine itself. In conjunction with the observation that 3-methyluridine is a very poor substrate compared to 5-methyluridine and 5,6-dimethyluridine, it follows that the phosphorolysis reaction involves the initial conversion of uridine, and other 5-substituted uridines (including 5-fluorouridine), to the syn conformation during interaction with the enzyme. Furthermore, and consistent with the foregoing, the enzyme recognizes as substrates, to varying degrees, the N(3)-ribosides of xanthine and uric acid, and will also catalyze the formation of these ribosides from the corresponding purines, which may be considered formally as 5,6-disubstituted uracils. Similar observations are reported for the synthetic 5,6-trimethyleneuridine. The enzyme does not, however, recognize 6-methyluracil and 5,6-tetramethyleneuridine in the reverse, synthetic, reaction. The conformational aspects of these reactions are discussed. Since it was previously shown that 6-methyluridine is an equally effective substrate for the pyrimidine phosphorylase of primary rabbit kidney cells, at least some of these conformational requirements apply to the enzyme from mammalian sources, and are consequently of relevance in the design of chemotherapeutic agents, for which some examples are cited.

Pyrimidine nucleoside analogues as inducers of pyrimidine nucleoside catabolizing enzymes in Salmonella typhimurium.

Various structural analogues of cytosine and uracil nucleosides were tested as potential inducers of the nucleoside catabolizing (cyt) enzymes in Salmonella typhimurium. Some analogues, e.g. 5'-O-alkyl cytidines and uridines, resistant to catabolic enzymes, were as effective as the natural inducers cytidine and uridine; but etherification of one of the cis 2' or 3'hydroxyls fully abolished activity, pointing to a requirement of an intact ribose cis-glycol system for activity. A uridine analogue in the syn conformation, 6-methyluridine, a good substrate for uridine phosphorylase, was inactive as an inducer. The behavior of various other analogues, in relation to their structure, conformation and substrate properties, indicated the absence of any correlation between inducing activity and substrate susceptibility. The overall findings are consistent with conclusions derived from genetic experiments. The active analogues apparently act via similar pathways, and probably affect the same regulatory mechanism(s) as the natural inducers.

Alkylated cytosine nucleosides: substrate and inhibitor properties in enzymatic deamination.

Cytosine nucleoside deaminase (EC 3.5.4.5) from Salmonella typhimurium LT2 catalyses the deamination of ribo-, deoxyribo- and arabinosyl nucleosides of cytosine alkylated at the C-5, but not at the N3 or exocyclic N4, of the pyrimidine ring. The enzyme was inert towards analogues etherified at the 3'-OH and 5'-OH of the sugar ring; it was active against the 2'-O-methyl derivative of cytidine, but not arabinosycytosine. The N4-and 5'-O-alkyl non-substrate analogues competitively inhibited deamination of deoxycytidine and arabinosylcytosine, the most inhibitory being 5'-O-methylarabinosylcytosine. The alpha anomer of 5'-ethyldeoxycytidine, the 2,2'-anhydro derivative of cytidine, and the 3'-O-alkyl derivatives were neither substrates nor inhibitors. The presence of cytidine deaminase was demonstrated in both granulocytes and lymphocytes from human peripheral blood. The specificity of this enzyme differed significantly from that of the bacterial enzyme, a finding of some relevance in relation to the frequently encountered intracellular deamination of therapeutically active arabinosylcytosine to the inactive arabinosyluracil.

Photochemical transformation of 5-alkyluracils and their nucleosides.

Irradiation at 254 nm of aqueous solutions of 5-ethyl-, 5-propyl-, and 5-isopropyluracils (or their nulcleosides) leads to cleavage of the 5-alkyl substituents, via an intramolecular electrocyclic photoaddition intermediate, with formation of uracil (or its nucleoside). The plhotoaddition intermediates represent a new class of dihydropyrimidines, namely analogs of 5,6-dihydro-5,6-cyclobutanyluracil and its nucleosides; the biological significance is discussed.