The Impact of Platelet Isolation Protocol on the Release of Extracellular Vesicles.

BACKGROUND: Platelet-derived extracellular vesicles (PEVs) are small vesicles released by activated platelets that are gaining growing interest in the field of vascular biology. The mode of platelet activation is a critical determinant of PEVs release, phenotype and function. However, only very limited information is available concerning the impact of the platelet purification procedure on PEVs release. METHODS: Washed or isolated platelets were separated by differential centrifugations. For washed platelets, the platelet pellet was washed by resuspension in PIPES buffer and finally resuspended in HEPES buffer. Isolated platelets were obtained by directly resuspending the platelet pellet in HEPES, skipping the washing steps in PIPES buffer. PEVs release was induced in washed or isolated platelets by stimulation with different agonist and analysed by Nanoparticle Tracking Analysis. RESULTS: Isolated platelets showed a higher release of PEVs upon adenosine diphosphate (ADP) stimulation compared to washed platelets, whereas PEVs released upon stimulation with strong agonists (thrombin, collagen, A23187, U46619) were similar in the two groups. This different responsiveness to ADP was also observed as a higher α-granules release and protein kinase C activation in isolated platelets compared to washed ones. Residual plasma contamination appeared to be essential for the ability of platelets to release PEVs in response to ADP. CONCLUSIONS: In conclusion, our study strongly suggests that procedure adopted for platelets preparation is a critical determinant of PEVs release upon ADP stimulation.

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase.

Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221 , with unit-cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

1H-NMR spectroscopy shows cellular uptake of HEPES buffer by human cell lines-an effect to be considered in cell culture experiments.

HEPES is commonly used in cell culture media as a buffering substance. Compared to the bicarbonate/CO2 buffer system, it does not require a CO2 atmosphere, thereby ensuring stable pH values during handling of cell culture media outside of an incubator. Due to its intrinsic charge, HEPES is considered not to be taken up by cells, which was a prerequisite during buffer development for cell culture by Good and colleagues. However, during the last years, evidence has emerged that HEPES seems to be taken up into cells and that it has major effects on cellular functions. Investigating three different cell lines (MCF-7, U2OS, HeLa) showed that all of them accommodated HEPES-containing medium, i.e., they survive and proliferate in the presence of HEPES. Determination of intracellular metabolites revealed the presence of HEPES for all cell lines. Further analysis of MCF-7 cells showed that even 48 h after medium exchange from HEPES-containing medium to HEPES-free medium, intracellular HEPES could still be detected. Thus, contrary to the common view, HEPES is taken up by cells which should be taken into consideration for studies of specific cellular functions. Graphical abstract ᅟ.

[Permeability of pannexin 1 channels to large anions].

It is widely accepted that ATP secretion in diverse cells involves pannexin 1 (Panx1) that functions as an ATP-permeable channel. We analyzed the permeability of Panx1 channels heterologically expressed in HEK-293 cells to a variety of anions, including ATP. As was demonstrated in electrophysiological experiments, relative permeabilities of studied species follow the sequence: C1- > MeSO4 > gluconate >> HEPES. This sequence suggests that ATP, which is more than twice as large as HEPES by mass, is most likely to be negligibly permeable to Panx1 channels. This inference was verified in experiments, where ATP secretion from Panx1-positive HEK-293 cells was assayed with the ATP-biosensor approach. It was shown that the heterologous expression of Panx1 in HEK-293, which normally are not ATP-secretive, did not endow transfected cells with the ability to liberate ATP in response to stimulation. Our data indicate that Panx1 alone forms anion channels with too low ATP permeability to mediate the secretory function. Nevertheless, the possibility still remains that certain ATP-permeable channels are heteromers of Panx1 and some other channel subunit(s).

Evaluation of resveratrol oxidation in vitro and the crucial role of bicarbonate ions.

Polyphenols can oxidize in culture medium and produce artifacts in cell culture studies. However, the extent and mechanism of the oxidation of resveratrol, a polyphenol abundant in red wine, is unclear. We investigated the oxidation of resveratrol in vitro and the effects of various components of the culture medium on the degradation of resveratrol and the production of H(2)O(2). We found that 96% of resveratrol at a concentration of 200 microM was degraded in Base Modified Eagle Medium after 24 h of incubation at 37 degrees C, producing about 90 microM of H(2)O(2). Including sodium bicarbonate in the medium markedly stimulated resveratrol degradation and H(2)O(2) production. In sum, we found that bicarbonate ions played a crucial role in the oxidative degradation of resveratrol in vitro, and that the degradation of resveratrol can be avoided by withdrawing sodium bicarbonate from the medium. A mechanism for the oxidation of resveratrol is proposed.

Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death.

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca(2+) chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca(2+) influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger, and KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca(2+ )stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca(2+ )release from intracellular Ca(2+ )stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis.

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin.

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.

3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects.

Crystal structure of bovine low molecular weight phosphotyrosyl phosphatase complexed with the transition state analog vanadate.

The early transition metal oxoanions vanadate, molybdate, and tungstate are widely used inhibitors for phosphatase enzymes. These oxoanions could inhibit such enzymes by simply mimicking the tetrahedral geometry of phosphate ion. However, in some cases, the enzyme-inhibitor dissociation constants (Ki) for these oxoanions are much lower than that for phosphate. Such observations gave rise to the hypothesis that in some cases these transition metal oxoanions may inhibit phosphomonoesterases by forming complexes that resemble the trigonal bipyramidal geometry of the SN2(P) transition state. As a test of this, the crystal structures of a low molecular weight protein tyrosine phosphatase at pH 7.5 complexed with the inhibitors vanadate and molybdate were solved at 2.2 A resolution and compared to a newly refined 1.9 A structure of the enzyme. Geometric restraints on the oxoanions were relaxed during refinement in order to minimize model bias. Both inhibitors were bound at the active site, and the overall protein structures were left unchanged, although some small but significant side chain movements at the active site were observed. Vanadate ion formed a covalent linkage with the nucleophile Cys12 at the active site and exhibited a trigonal bipyramidal geometry. In contrast, simple tetrahedral geometry was observed for the weaker molybdate complex. These studies are consistent with the conclusion that vanadate inhibits tyrosine phosphatases by acting as a transition state analog. The structure of the vanadate complex may be expected to closely resemble the transition state for reactions catalyzed by protein tyrosine phosphatases.

In vitro formation of activators for prostaglandin synthesis by neutrophils and macrophages from humans and guinea pigs.

Prostaglandin H synthase, the primary enzyme in the pathway to the prostaglandins, requires the continued presence of a hydroperoxide activator for its enzyme activity. Phagocytic leukocytes from either humans or guinea pigs produced activator hydroperoxides in quantities sufficient to enhance prostaglandin synthesis in cells. Compounds that stimulated the oxidative burst (e.g., phorbol myristate acetate, opsonized zymosan, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine) enhanced the overall production of the activators. Accumulation of activator(s) was promoted by exogenous Fe+3 (2 mumol/L), adenosine diphosphate (10 mumol/L), and unsaturated fatty acids (1 to 30 mumol/L) and was completely inhibited by glutathione peroxidase (0.5 U/ml). Catalase (500 U/ml) decreased the amount of activator by 70% when added during the incubation but by only 40% when added after the incubation. Thus, the activator appeared to be partly H2O2 and partly a lipid hydroperoxide. The addition of H2O2 in quantities similar to those produced by phagocytes increased prostaglandin formation by twofold in incubations with U937 cells and carbon 14-labeled arachidonic acid (2 mumol/L). These results indicate a new role for the oxygen metabolites from leukocytes in providing an intercellular signal that can stimulate prostaglandin synthesis.