The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. Not only homopolymeric poly(A)-tails, comprised exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. Polynucleotide phosphorylase (PNPase) and the archaeal exosome, which bear strong similarities to one another, both functionally and structurally, were found to polymerize the heteropolymeric tails in bacteria, spinach chloroplasts, and archaea. As phosphorylases, these enzymes use diphosphate nucleotides as substrates and can reversibly polymerize or degrade RNA, depending on the relative concentrations of nucleotides and inorganic phosphate. A possible scenario, illustrating the evolution of RNA polyadenylation and its related functions, is presented, in which PNPase (or the archaeal exosome) was the first polyadenylating enzyme to evolve and the heteropolymeric tails that it produced, functioned in a polyadenylation-stimulated RNA degradation pathway. Only at a later stage in evolution, did the poly(A)-polymerases that use only ATP as a substrate, hence producing homopolymeric adenosine extensions, arise. Following the appearance of homopolymeric tails, a new role for polyadenylation evolved; RNA stability. This was accomplished by utilizing stable poly(A)-tails associated with the mature 3' ends of transcripts. Today, stable polyadenylation coexists with unstable heteropolymeric and homopolymeric tails. Therefore, the heteropolymeric poly(A)-rich tails, observed in bacteria, organelles, archaea, and human cells, represent an ancestral stage in the evolution of polyadenylation.

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.

Extracellular ATP regulates proliferation and differentiation, functioning as an important messenger via purinergic (P2) receptors in keratinocytes. In this study, we investigated the effects of ATP on cytokine production in cultured normal human epidermal keratinocytes (NHEKs). Adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), adenosine 5'-O-2-(thio)diphosphate (ADPbetaS), ADP, ATP, and 2', 3'-O-(4-benzoyl-benzoyl) ATP (BzATP) significantly increased the release of IL-6. The P2 antagonists, suramin-, reactive blue 2-, and periodate-oxidized ATP, inhibited ATP-induced IL-6 release, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, adenosine 3'-phosphate 5'-phosphate, 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, and pertussis toxin did not. SQ22563, an adenylate cyclase inhibitor, inhibited ATP-induced IL-6 release. ATPgammaS, ADPbetaS, ATP, and BzATP significantly increased the intracellular cAMP content. Reverse transcription-PCR showed expression of P2Y1, P2Y2, P2Y4, P2Y11, P2Y12, P2Y13, P2X1, P2X4, P2X5, P2X6, and P2X7 receptor subtypes. Additionally, UVB radiation evoked the release of ATP from NHEKs. The release of IL-6 and the expression of IL-6 mRNA were increased after UVB radiation, and these increases were also inhibited by P2 receptor antagonists. These results suggest that cAMP-generating P2Y receptors are likely functional in ATP-induced IL-6 production in NHEKs. Furthermore, in UVB-radiated cells, we note the possibility that P2 receptor antagonists may reduce skin inflammation.

BACKGROUND

In this study, we tested the ability of a novel poly(adenosine diphosphate ribose) polymerase (PARP) inhibitor, 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]-anthracen-3-one (GPI-15427), to enhance the effect of radiotherapy in a xenograft model of human head and neck squamous cell carcinoma (HNSCC).

METHODS

Human xenograft HNSCC tumors were established in female nude mice: animals were treated with orally administered GPI-15427 at varied doses prior to tumor irradiation. In vitro and in vivo apoptosis analyses and neutral single-cell gel electrophoresis (comet) assay were performed, with the "tail moment" calculated to evaluate DNA double-strand break damage.

RESULTS

Orally administered GPI-15427 given before radiation therapy significantly reduced tumor volume, and cells demonstrated significantly elevated mean tail moments (indicative of DNA damage) and enhanced apoptosis both in vitro and in vivo, compared with radiation-alone and control groups.

CONCLUSIONS

Use of the PARP-1 inhibitor GPI-15427 induced significant sensitization to radiotherapy, representing a promising new treatment in the management of HNSCC.

Mitochondria isolated from kidneys of lead-intoxicated rats have been shown to have decreased oxidative and phosphorylative abilities. The purpose of this study was to determine whether these abnormal mitochondria would undergo ultrastructural transformation during controlled respiration in the absence of phosphate acceptor (State IV), as previously demonstrated for normal liver mitochondria. It was first shown that normal rat kidney mitochondria transforms from a condensed ultrastructural conformation to an orthodox conformation after 5 min of State IV respiration with pyruvate-malate substrate. Reversal to a condensed conformation follows stimulation of respiration with adenosine diphosphate (ADP). A large portion of kidney mitochondria from lead-poisoned rats do not change from condensed to orthodox conformation during State IV respiration. Other mitochondria do transform to the orthodox form but they rapidly degenerate. State IV respiration decreases as these few orthodox mitochondria disintegrate. The conclusion is that those mitochondria that do not undergo change in ultrastructure have impairment of electron transport, and that those that do become orthodox have increased membrane lability and undergo degeneration.

Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The K(m) for ATP of this enzyme is 5.5 x 10(-4)m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg(++) and very slightly stimulated by Ca(++). The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na(+) nor K(+) ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10(-4)m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria.

Multiple mutations in the gene responsible for cystic fibrosis are located within a region predicted to encode a nucleotide-binding fold in the amino terminal half of the cystic fibrosis transmembrane conductance regulator protein. A 67-amino acid peptide (P-67) that corresponds to the central region of this putative nucleotide binding site was chemically synthesized and purified. This peptide bound adenine nucleotides. The apparent dissociation constants (Kd's) for the trinitrophenyl (TNP) adenine nucleotides, TNP-adenosine triphosphate, TNP-adenosine diphosphate, and TNP-adenosine monophosphate, were 300 nanomolar, 200 nanomolar, and greater than 1 micromolar, respectively. The Kd for adenosine triphosphate was 300 micromolar. Circular dichroism spectroscopy was used to show that P-67 assumes a predominantly beta sheet structure in solution, a finding that is consistent with secondary structure predictions. On the basis of this information, the phenylalanine at position 508, which is deleted in approximately 70 percent of individuals with cystic fibrosis, was localized to a beta strand within the nucleotide binding peptide. Deletion of this residue is predicted to induce a significant structural change in the beta strand and altered nucleotide binding.

Platelet function was studied before and 1 hour after ingestion of 20 mg nifedipine, a new calcium antagonist, in 20 patients with coronary heart disease. Platelet counts remained unchanged. Platelet adhesiveness, measured as retention in glass bead columns with Hellem's method for native blood, did not drop significantly eigher when 0.9 or 3.6 ml of blood was used. Platelet aggregation, which is dependent on extracellular calcium, was induced in citrated platelet-rich plasma. The mean maximal rate of primary aggregation, initiated with three different concentrations of adenosine diphosphate, was reduced by 20% to 26%. The rate of irreversible collagen-induced aggregation was on average 23% lower after nifedipine. The mean bleeding time was 36 seconds, or 12%, longer after ingestion of the drug. The moderate, but significant reduction of platelet aggregation and prolongation of the bleeding time by nifedipine may be mediated through inhibition of calcium transport across the platelet membrane.

Platelets play roles in both thrombosis and inflammation, and chemokines that are released at sites of inflammation could potentially activate platelets. Among the chemokine receptors expressed on platelets, the CXCR4 is the receptor for chemokine stromal cell-derived factor-1 (SDF-1), and the CCR4 is the receptor for macrophage-derived chemokine (MDC). Of the chemokines tested, SDF-1 and MDC were the only 2 that activated platelets. Both are weak agonists, but they enhanced response to low-dose adenosine 5'-diphosphate (ADP), epinephrine, or serotonin. When SDF-1 and MDC were added together, full and brisk platelet aggregation occurred. Platelet activation by these 2 chemokines appears to involve distinct pathways: SDF-1 inhibited an increase in cyclic adenosine monophosphate (cAMP) following prostaglandin (PG) I(2), while MDC had no effect. In contrast, MDC, but not SDF-1, lead to Ca(++) mobilization by platelets. Further, second-wave aggregation induced by MDC in platelet-rich plasma was inhibited by aspirin, ADP scavenger creatine phosphate/creative phosphokinase (CP/CPK), and ARL-66096, an antagonist of the ADP P2T(AC) receptor involved in adenylyl cyclase inhibition. But the aggregation was not affected by A3P5PS, an inhibitor of the ADP P2Y receptor. SDF-1-induced aggregation was inhibited by aspirin, but it was only slightly affected by CP/CPK, ARL-66096, or A3P5PS. Finally, the presence of chemokines in platelets was determined. Reverse transcriptase-polymerase chain reaction studies with platelet RNA did not detect the presence of SDF-1 or MDC. In summary, SDF-1 and MDC are platelet agonists that activate distinct intracellular pathways. Their importance in the development of thrombosis at sites of inflammation needs to be further evaluated.

Members of the Src family of kinases are abundant in platelets. Although their localization is known, their role(s) in platelet function are not well understood. Lyn is a Src-family kinase that participates in signal transduction pathways elicited by collagen-related peptide; it has also been implicated through biochemical studies in the regulation of von Willebrand factor signaling. Here, we provide evidence that Lyn plays a role in gamma-thrombin activation of platelets. Unlike the wild-type platelets, platelets from Lyn-deficient mice do not undergo irreversible aggregation, produce thromboxane A2, or secrete adenosine diphosphate in response to submaximal gamma-thrombin concentrations that cause secretion-dependent irreversible aggregation. Phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase, also requires a higher concentration of gamma-thrombin in Lyn-deficient platelets than in wild-type platelets. These findings demonstrate that Lyn signaling is required for thrombin induction of secretion-dependent platelet aggregation. Specifically, Lyn is required under these conditions to enable thrombin-induced TxA2 production and adenosine diphosphate secretion, necessary steps in secretion-dependent platelet aggregation.

OBJECTIVE

To examine the associations between air temperature and risk factors for ischaemic heart disease.

METHODS

Data on risk factors are available from up to 2036 men in the Caerphilly Prospective Heart Disease Study. Daily temperatures were obtained from the Meteorological Office. Relations between these were examined by regression.

RESULTS

The coldest month of the year has a mean temperature that is 16 degrees C lower than that in the warmest month. A fall in temperature of this magnitude is associated with higher blood pressures (by 3-5 mm Hg) and a lower concentration of high density lipoprotein cholesterol (by 0.08 mmol/l). The most important effects however, seem to be on the haemostatic system. Fibrinogen is 0.34 g/l higher in the coldest month than in the warmest (p < 0.001) and alpha 2 macroglobulin, a protein that inhibits fibrinolysis, is also raised. Platelet count is increased by 30% of a standard deviation and the sensitivity of platelets in whole blood to adenosine diphosphate is increased by cold.

CONCLUSIONS

These effects on haemostasis, together with the effect on blood pressure, could explain a large part of the increase in ischaemic heart disease in the winter but are unlikely to explain much of the difference in mortality within different areas of England and Wales.

Drug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7, -8, -10, and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) in cells from children with acute lymphoblastic leukemia (ALL; n = 43) and acute myeloid leukemia (AML; n = 10). PARP expression was present in all B-lineage samples, but absent in 4 of 15 T-lineage ALL samples and 3 of 10 AML cases, which was not caused by genomic deletions. PARP expression was a median 7-fold lower in T-lineage ALL (P < .001) and 10-fold lower in AML (P < .001) compared with B-lineage ALL. PARP expression was 4-fold lower in prednisolone, vincristine and L-asparaginase (PVA)-resistant compared with PVA-sensitive ALL patients (P < .001). Procaspase-2 expression was 3-fold lower in T-lineage ALL (P = .022) and AML (P = .014) compared with B-lineage ALL. In addition, procaspase-2 expression was 2-fold lower in PVA-resistant compared to PVA-sensitive ALL patients (P = .042). No relation between apoptotic protease-activating factor 1 (Apaf-1), procaspases-3, -6, -7, -8, -10, and drug resistance was found. In conclusion, low baseline expression of PARP and procaspase-2 is related to cellular drug resistance in childhood acute lymphoblastic leukemia.

The brain's energy demands are remarkable both in their intensity and in their moment-to-moment dynamic range. This perspective considers the evidence for Warburg-like aerobic glycolysis during the transient metabolic response of the brain to acute activation, and it particularly addresses the cellular mechanisms that underlie this metabolic response. The temporary uncoupling between glycolysis and oxidative phosphorylation led to the proposal of an astrocyte-to-neuron lactate shuttle whereby during stimulation, lactate produced by increased glycolysis in astrocytes is taken up by neurons as their primary energy source. However, direct evidence for this idea is lacking, and evidence rather supports that neurons have the capacity to increase their own glycolysis in response to stimulation; furthermore, neurons may export rather than import lactate in response to stimulation. The possible cellular mechanisms for invoking metabolic resupply of energy in neurons are also discussed, in particular the roles of feedback signaling via adenosine diphosphate and feedforward signaling by calcium ions.

BACKGROUND

Two point-of-care (POC) systems have been recently proposed as rapid tools with which to evaluate residual platelet reactivity (RPR) in coronary artery disease (CAD) patients.

OBJECTIVE

We compared Platelet Function Analyzer-100 (PFA-100) closure times (CTs) by collagen/adenosine 5'-diphosphate (ADP) (C/ADP CT) cartridge and the VerifyNow P2Y12 Assay (VerifyNow) with light transmission aggregation (LTA) induced by 2 and 10 micromol L(-1) ADP in 1267 CAD patients on dual antiplatelet therapy who underwent percutaneous coronary intervention. We also performed the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay by cytofluorimetric analysis in a subgroup of 115 patients.

RESULTS

Cut-off values for identifying RPR were:>> or = 54% and>> or = 66% for LTA induced by 2 and 10 micromol L(-1) ADP respectively, and>> or = 264 P2Y12 Reaction Units (PRU) for VerifyNow. The cut-off for PFA-100 C/ADP CT was>> or = 68 s. RPR was detected in 25.1% of patients by 2 mumol L(-1) ADP-induced LTA (ADP-LTA), in 23.2% by 10 micromol L(-1) ADP-LTA, in 24.4% by PFA-100, and in 24.7% by VerifyNow. PFA-100 results did not parallel those obtained with LTA. VerifyNow showed a significant correlation (rho = 0.62, P < 0.001) and significant agreement (k = 0.34, P < 0.001) with LTA induced by 2 micromol L(-1) ADP. The correlation was similar but the agreement was better between VerifyNow and 10 micromol L(-1) ADP-LTA (rho = 0.64, P < 0.0001; k = 0.43, P < 0.001). Significant relationships were found between VASP platelet reactivity index and both ADP-LTA and VerifyNow. PFA-100 C/ADP CT did not significantly correlate with any of the other assays.

CONCLUSIONS

Our results show a significant correlation between LTA and VerifyNow but not the PFA-100 C/ADP assay. Clinical validation studies for POC systems are necessary.

Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5'-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with (3)H-labelled nucleotides and adenosine as substrates, direct evaluation of gamma-phosphate transfer from [gamma-(32)P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20 kDa surface protein was observed following incubation of Namalwa B cells with [gamma-(32)P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.

BACKGROUND

SRT2104 has been developed as a selective small molecule activator of SIRT1, a NAD(+)-dependent deacetylase involved in the regulation of energy homeostasis and the modulation of various metabolic pathways, including glucose metabolism, oxidative stress and lipid metabolism. SIRT1 has been suggested as putative therapeutic target in multiple age-related diseases including type 2 diabetes and dyslipidemias. We report the first clinical trial of SRT2104 in elderly volunteers.

METHODS

Oral doses of 0.5 or 2.0 g SRT2104 or matching placebo were administered once daily for 28 days. Pharmacokinetic samples were collected through 24 hours post-dose on days 1 and 28. Multiple pharmacodynamic endpoints were explored with oral glucose tolerance tests (OGTT), serum lipid profiles, magnetic resonance imaging (MRI) for assessment of whole body visceral and subcutaneous fat, maximal aerobic capacity test and muscle 31P magnetic resonance spectroscopy (MRS) for estimation of mitochondrial oxidative capacity.

RESULTS

SRT2104 was generally safe and well tolerated. Pharmacokinetic exposure increased less than dose-proportionally. Mean Tmax was 2-4 hours with elimination half-life of 15-20 hours. Serum cholesterol, LDL levels and triglycerides decreased with treatment. No significant changes in OGTT responses were observed. 31P MRS showed trends for more rapid calculated adenosine diphosphate (ADP) and phosphocreatine (PCr) recoveries after exercise, consistent with increased mitochondrial oxidative phosphorylation.

CONCLUSIONS

SRT2104 can be safely administered in elderly individuals and has biological effects in humans that are consistent with SIRT1 activation. The results of this study support further development of SRT2104 and may be useful in dose selection for future clinical trials in patients.

BACKGROUND

ClinicalTrials.gov NCT00964340.

Ecto-5'-nucleotidase (CD73) is an essential enzyme that generates adenosine, an essential molecule for cell growth. CD73 increases significantly in many breast cancers. In this study, alpha,beta-methylene adenosine-5'-diphosphate (APCP), a specific CD73 inhibitor was used to block the hydrolase's activity. Effects of CD73 were examined on human breast cancer cells MDA-MB-231 in culture for proliferation, cell cycle progression, and apoptosis before and after APCP treatment. The in vivo effect of CD73 was examined on MDA-MB-231 tumor xenograft growth in nude mice. Cell growth curve, cell cycle and apoptosis were observed with MTT assays and flow cytometry, respectively. Microvessel density (MVD) and lymph vessel density (LVD) of implanted tumor tissues was analyzed by immunohistochemistry for CD31 and VEGFR-3 staining respectively. Our results showed that APCP inhibited MDA-MB-231 viability in a dose-dependent manner. APCP (12 microM) increased the percentage of G0/G1 phase cells from 49.75 to 59.16% while it decreased S phase and G2/M cells from 24.85 and 18.65% to 21.65 and 12.55%, respectively. The percentages of early and late apoptotic cells were also decreased after APCP treatment. However, APCP treatment did not affect the percentage of normal cells. Xenograft of MDA-MB-231 cells in the APCP treatment group had lower volume and weight than those of control group (2.70+/-1.14 vs 1.41+/-0.39 cm(3) and 2.7+/-0.5 vs 1.3+/-0.2 g), accompanied with less vessel formation with a MVD of 5+/-1 compared to the control group's 10+/-2 and an LVD of 4+1 vs 7+2. Our results suggest that CD73 may promote tumor growth and serve as a marker of breast cancer progression.

OBJECTIVE

Body temperature may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. In addition, mild hypothermia is used to treat intracranial hypertension in human liver failure without detailed information on its effects on the injured liver itself. Therefore, we investigated the effects of body temperature on the progression of APAP-induced liver injury in mice.

METHODS

Male C57BL6 mice treated with saline or APAP (300 mg/kg intraperitoneally) were maintained at normothermia (35.5-37.5 degrees C) by external warming or were allowed to develop mild hypothermia (32.0-35.0 degrees C) after 2 hours from APAP administration.

RESULTS

Mild hypothermia resulted in improved survival after APAP intoxication. Liver damage was reduced, as assessed histologically and by plasma alanine aminotransferase levels. Early effects of hypothermia included a reduction of hepatic congestion and improved recovery of glycogen stores. At later time points (8-12 hours), APAP-treated mice that were maintained at normothermia manifested increased hepatocyte apoptosis, as assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining and cleavage of poly(adenosine diphosphate-ribose) polymerase. Mild hypothermia did not affect the formation of APAP-protein adducts or the depletion of glutathione, nor did it abrogate hepatocyte DNA synthesis.

CONCLUSIONS

Mild hypothermia improved survival and attenuated liver injury and apoptosis in APAP-treated mice by reducing hepatic congestion and improving glycogen recovery without affecting hepatic regeneration. Results of the study underscore the need for a strict control of body temperature in animal models of liver failure and suggest that the benefits of mild hypothermia in liver failure may extend beyond those related to reduced cerebral complications.

OBJECTIVE

We investigated the role of P-selectin in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates.

BACKGROUND

Plaque rupture followed by thrombus formation is a fundamental pathophysiology of acute coronary syndromes. Although the adhesive interaction between platelets and leukocytes via P-selectin is known to mediate platelet-rich thrombi, the true function of P-selectin in thrombus formation in vivo is unknown.

METHODS

In wild-type (P(+/+)) and P-selectin-deficient (P(-/-)) mice with ferric chloride (FeCl(3))-induced carotid arterial thrombosis model, we measured in vivo platelet P-selectin expression and adenosine diphosphate (ADP)-induced ex vivo platelet aggregation. We also measured ex vivo ADP-induced whole blood aggregations and their size distribution by flow cytometry.

RESULTS

Time to thrombotic occlusion was longer in P(-/-) mice than in P(+/+) mice. Spontaneous reflow after total thrombotic occlusion was observed in 8 of 10 P(-/-) mice but not in any P(+/+) mice. ADP-induced ex vivo platelet aggregation was not different between the two groups. However, ADP-induced ex vivo whole blood aggregation was inhibited in P(-/-) mice compared to P(+/+) mice. FeCl(3) application increased in vivo expressions of platelet P-selectin in P(+/+) mice but not in P(-/-) mice. The number of leukocytes within thrombi was less in P(-/-) mice than in P(+/+) mice. In flow cytometric analysis of size distribution of ADP-induced whole blood aggregates, the number of large aggregates was less in P(-/-) mice than in P(+/+) mice. Using platelet and leukocyte fluorescence makers, the large aggregates were confirmed as platelet-leukocyte aggregates.

CONCLUSIONS

Platelet P-selectin plays an important role in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates.

OBJECTIVE

Trichostatin A (TSA) is a histone deacetylase inhibitor that causes growth inhibition of malignant cells. The authors' goal was to evaluate its effect on cell growth and cell cycle regulation in a large panel of glioma cell lines, as well as in human astrocytes, fibroblasts, and endothelial cells.

METHODS

Cell growth in response to TSA was evaluated using a tetrazolium colorimetric assay and a clonogenic assay. Cell cycle effects were examined using flow cytometry. A DNA fragmentation assay was used to evaluate induction of apoptosis. Histone acetylation status and the expression of p21WAF1, phosphorylated retinoblastoma protein (Rb), poly(adenosine diphosphate-ribose) polymerase (PARP), and caspase-3 were studied using Western blot analysis. In the glioma cell lines, there was significant inhibition of cell growth and detection of increased levels of acetylated histones after TSA treatment. The mechanisms underlying the growth inhibition include cell cycle arrest at the G2/M phase and apoptosis induction. The expression of p21WAF1 was activated, with a temporally related decrease in levels of phosphorylated Rb. Apoptosis was preceded by detection of cleaved PARP and activated caspase-3. The effects of TSA were less pronounced or absent in human astrocytes, fibroblasts, and endothelial cells.

CONCLUSIONS

The TSA caused inhibition of glioma cell growth by both cell cycle arrest and apoptosis. Cell cycle arrest was associated with an increase in p21WAF1 expression and a decrease in phosphorylated Rb. Apoptosis was mediated at least partly through the activation of caspase-3. Because of the differential effects in glioma cells compared with nonneoplastic cells, TSA may provide a novel strategy for achieving tumor growth inhibition and cytotoxicity. Further investigation is warranted.

In elite outfield players, the average work rate during a soccer match, as estimated from variables such as heart rate, is approximately 70% of maximal oxygen uptake (VO2 max). This corresponds to an energy production of approximately 5700 kJ (1360 kcal) for a person weighing 75 kg with a VO2 max of 60 ml kg-1 min-1. Aerobic energy production appears to account for more than 90% of total energy consumption. Nevertheless, anaerobic energy production plays an essential role during soccer matches. During intensive exercise periods of a game, creatine phosphate, and to a lesser extent the stored adenosine triphosphate, are utilized. Both compounds are partly restored during a subsequent prolonged rest period. In blood samples taken after top-class soccer matches, the lactate concentration averages 3-9 mM, and individual values frequently exceed 10 mM during match-play. Furthermore, the adenosine diphosphate degradation products--ammonia/ammonium, hypoxanthine and uric acid--are elevated in the blood during soccer matches. Thus, the anaerobic energy systems are heavily taxes during periods of match-play. Glycogen in the working muscle seems to be the most important substrate for energy production during soccer matches. However, muscle triglycerides, blood free fatty acids and glucose are also used as substrates for oxidative metabolism in the muscles.

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).

The platelet-rich plasma (PRP) is an autologous biotherapy based on platelet-healing properties. Here, we developed a simple and reproducible PRP purification protocol based on two successive centrifugations. We evaluated different centrifugation speeds and time-storage durations on the platelet quantity and quality. Sterility and stability of our PRP homemade product were also performed. We prepared PRP from 54 healthy volunteers. We tested activation state, reactivity, and stability of platelets by flow cytometry using basal and adenosine diphosphate (ADP)-induced P-selectin expression markers; growth factor release after platelet activation by an enzyme-linked immunosorbent assay (ELISA); platelet aggregation capacity by aggregrometry assays; clot formation and retraction by thromboelastography; and platelet morphology by ultrastructural analysis. About 130 and 250 g successive speed centrifugations further concentrated platelets while preserving their bioactivity during 6 h (after that, platelet functions were significantly altered). In these conditions, we obtained a highly concentrated pure PRP product (with a low leukocyte count) suitable to study platelet properties. To avoid the loss of efficacy, we recommend injecting PRP under 3 h after preparation.