The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.

Within the skeletal muscle cell at the onset of muscular contraction, phosphocreatine (PCr) represents the most immediate reserve for the rephosphorylation of adenosine triphosphate (ATP). As a result, its concentration can be reduced to less than 30% of resting levels during intense exercise. As a fall in the level of PCr appears to adversely affect muscle contraction, and therefore power output in a subsequent bout, maximising the rate of PCr resynthesis during a brief recovery period will be of benefit to an athlete involved in activities which demand intermittent exercise. Although this resynthesis process simply involves the rephosphorylation of creatine by aerobically produced ATP (with the release of protons), it has both a fast and slow component, each proceeding at a rate that is controlled by different components of the creatine kinase equilibrium. The initial fast phase appears to proceed at a rate independent of muscle pH. Instead, its rate appears to be controlled by adenosine diphosphate (ADP) levels; either directly through its free cytosolic concentration, or indirectly, through its effect on the free energy of ATP hydrolysis. Once this fast phase of recovery is complete, there is a secondary slower phase that appears almost certainly rate-dependent on the return of the muscle cell to homeostatic intracellular pH. Given the importance of oxidative phosphorylation in this resynthesis process, those individuals with an elevated aerobic power should be able to resynthesise PCr at a more rapid rate than their sedentary counterparts. However, results from studies that have used phosphorus nuclear magnetic resonance ((31)P-NMR) spectroscopy, have been somewhat inconsistent with respect to the relationship between aerobic power and PCr recovery following intense exercise. Because of the methodological constraints that appear to have limited a number of these studies, further research in this area is warranted.

We studied brain and muscle energy metabolism by phosphorus 31 magnetic resonance spectroscopy (31P-MRS) in 12 patients affected by migraine with aura (classic migraine) in interictal periods. Brain 31P-MRS disclosed a low phosphocreatine content in all patients, accompanied by high adenosine diphosphate concentration, a high percentage of V/Vmax (adenosine triphosphate), and a low phosphorylation potential--features showing an unstable state of metabolism in classic migraine. Abnormal muscle mitochondrial function, in the absence of clinical signs of muscle impairment, was present in nine of the 12 patients examined.

Treatment of thrombotic disease requires a delicate balance between prevention of new thrombotic events and management of bleeding complications. Various antiplatelet and anticoagulant agents have been used to this end, with varying degrees of success. Among the antiplatelet agents tested so far, cilostazol, which selectively targets phosphodiesterase III (PDE-III), has unique features. Cilostazol is classified as an antiplatelet agent because it inhibits the platelet aggregation induced by collagen, 5'-adenosine diphosphate (ADP), epinephrine, and arachidonic acid. Unlike other antiplatelet agents cilostazol not only inhibits platelet function but also improves endothelial cell function. Platelets circulate throughout the body with continuous tethering on the surface of endothelial cells. When endothelial cells are stimulated, the number of activated, or pre-conditioned, platelets in circulation increases. These platelets enhance thrombus formation at the sites of endothelial disruption. Cilostazol effectively prevents the onset of thrombotic disease, not only by direct inhibition of platelet function, but also by reducing the number of activated or pre-conditioned platelets in circulation. Secondary prevention of stroke with modest increase in bleeding complications was achieved by administration of cilostazol in the Japanese Cilostazol Stroke Prevention Study done in Japan. These results suggest that cilostazol may reduce the risk of stroke without increasing the risk of bleeding complications.

Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, FcgammaRIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes (ACS) as compared with SAP or C (C, 51.2+/-12.6%; SAP, 44.9+/-13.6%; UAP, 28.4+/-10.1%; AMI, 20.3+/-8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients' sera revealed significantly elevated concentrations of tumor necrosis factor alpha, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1beta in ACS (vs. C and SAP). IFN-gamma, GM-CSF, and IL-1beta significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5'-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-gamma, GM-CSF, and IL-1beta and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS.

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However, little is known about the biological characteristics of recurrent HCC cells. A single patient's primary and recurrent HCC-derived cell lines, Hep-11 and Hep-12, respectively, were established by primary culture. These two cell lines have the same hepatitis B virus integration site and share many common amplifications and deletions, which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10 000 cells, injection of only 100 Hep-12 cells was sufficient to initiate tumor growth, and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11, Hep-12 cells expressed the oval cell markers AFP, NCAM/CD56, c-kit/CD117, as well as multiple stem cell markers such as Nanog, OCT4 and SOX2. In addition, >90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel, but more resistant to doxorubicin, cisplatin and hydroxycamptothecin (HCPT), which had been administrated to the patient. Furthermore, Hep-12 cells expressed higher levels of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) than Hep-11, and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 cells but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells and that elevated expression of PARP-1 was related to their resistance to HCPT.

Metastasis is a multistep process by which tumor cells disseminate from their primary site and form secondary tumors at a distant site. The pathophysiological course of metastasis is mediated by the dynamic plasticity of cancer cells, which enables them to shift between epithelial and mesenchymal phenotypes through a transcriptionally regulated program termed epithelial-to-mesenchymal transition (EMT) and its reverse process, mesenchymal-to-epithelial transition (MET). Using a mouse model of spontaneous metastatic breast cancer, we investigated the molecular mediators of metastatic competence within a heterogeneous primary tumor and how these cells then manipulated their epithelial-mesenchymal plasticity during the metastatic process. We isolated cells from the primary mammary tumor, the circulation, and metastatic lesions in the lung in TA2 mice and found that the long noncoding RNA (lncRNA) H19 mediated EMT and MET by differentially acting as a sponge for the microRNAs miR-200b/c and let-7b. We found that this ability enabled H19 to modulate the expression of the microRNA targets Git2 and Cyth3, respectively, which encode regulators of the RAS superfamily member adenosine 5'-diphosphate (ADP) ribosylation factor (ARF), a guanosine triphosphatase (GTPase) that promotes cell migration associated with EMT and disseminating tumor cells. Decreasing the abundance of H19 or manipulating that of members in its axis prevented metastasis from grafts in syngeneic mice. Abundance of H19, GIT2, and CYTH3 in patient samples further suggests that H19 might be exploited as a biomarker for metastatic cells within breast tumors and perhaps as a therapeutic target to prevent metastasis.

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site.

In an earlier report on the kidney in systemic lupus erythematosus (SLE), we described a subset of patients with circulating anticoagulants; many had glomerular and arteriolar thrombosis in the absence of necrosis and subendothelial deposits. The present study extends these observations to a larger group of patients with SLE and a circulating anticoagulant, and compares its findings with those in patients with SLE without evidence of an anticoagulant. It demonstrates (1) a higher prevalence of clinically recognizable thrombotic events in the venous and arterial circulations in patients with SLE and a detectable anticoagulant; (2) a probable shortening in life span; (3) a higher prevalence of glomerular thrombi; (4) elevated levels of factor VIII antigen and von Willebrand factor; and (5) significantly lower platelet counts and decreased in vitro platelet aggregation in response to adenosine diphosphate, epinephrine, and collagen. Since prednisone treatment often results in improvement or disappearance of a prolonged partial thromboplastin time, the test most commonly used for screening of a circulating anticoagulant, we suggest that the prevalence of this abnormality may be underestimated in patients with SLE.

We studied the pharmacokinetic and pharmacodynamic properties of integrelin, a novel platelet glycoprotein IIb/IIIa receptor inhibitor, in patients undergoing elective percutaneous coronary intervention. Patients were randomized to placebo (n = 19) or to 1 of 4 integrelin dosing regimens (total n = 54) that were studied sequentially. All patients received aspirin and heparin. Patients were followed until discharge for the occurrence of adverse clinical events: death, myocardial infarction, coronary artery bypass surgery, repeat intervention, or recurrent ischemia. Bleeding was the primary safety end point. Frequent blood sampling was performed for adenosine diphosphate-induced platelet aggregations. Simplate bleeding times were performed. Adverse clinical events occurred less often in the integrelin-treated patients, although the overall numbers were too small to make a definitive statement as to clinical efficacy. There was no significant increase in serious bleeding among integrelin-treated patients. The 2 highest integrelin boluses (180 and 135 micrograms/kg) immediately (15 minutes after the bolus) provided>> 80% inhibition of adenosine diphosphate-induced platelet aggregation in>> 75% of treated patients. A constant integrelin infusion of 0.75 micrograms/kg/min maintained this marked antiplatelet effect, whereas an infusion of 0.50 micrograms/kg/min allowed gradual recovery of platelet function. Elective coronary intervention was performed safely and with no significant increase in serious bleeding events using integrelin with aspirin and heparin as an antithrombotic regimen. Integrelin provided rapid, intense, and persistent ex vivo platelet inhibition during coronary intervention. This new antiplatelet agent may be beneficial in reducing platelet-mediated ischemic complications of percutaneous coronary intervention.

To test the ability of regenerated endothelium to evoke endothelium-dependent relaxations, male Yorkshire pigs underwent balloon endothelial denudation of the proximal left anterior descending coronary artery. Endothelium-dependent responses were examined in vitro, in rings of coronary segments taken from the denuded area or from the proximal left circumflex coronary artery. The experiments were performed 8 days or 4 weeks after the denudation. Endothelial regrowth was confirmed by histologic examination 8 days after the denudation and by demonstrating the presence of endothelium-dependent relaxations to bradykinin; at that time aggregating platelets evoked normal endothelium-dependent responses. However, 4 weeks after the denudation, the relaxations to aggregating platelets were markedly depressed although continuous endothelial lining was present, and the endothelium-dependent responses to bradykinin, adenosine diphosphate, the Ca2+-ionophore A23187, platelet activating factor, and thrombin were unaltered. Four weeks after denudation, endothelium-dependent relaxations to serotonin were depressed. Higher concentration of serotonin induced endothelium-dependent contractions in quiescent rings with regenerated endothelium, suggesting that regenerated endothelial cells may produce endothelium-derived constricting factor(s) and release less endothelium-derived relaxing factor(s) when exposed to the monoamine. The endothelium-dependent relaxation to serotonin was not reduced by the S2-serotonergic antagonist ketanserin but prevented by the combined S1- and S2-serotonergic blocker methiothepin. The platelet-induced relaxation was due to released serotonin and adenine nucleotides in control left circumflex coronary arteries, but in left anterior descending coronary artery with regenerated endothelium, it was due solely to the latter. The platelet-induced contractions were due to activation of receptors on the smooth muscle cells. Four weeks after denudation, regenerated endothelial cells were morphologically different from native cells; they were elongated and cuboidal, and the number of the cells had increased twofold. At this state, eccentric myointimal thickening was present in the previously denuded portion. These experiments indicate that the protective role of endothelial cells against the vasoconstriction induced by aggregating platelets is depressed in the chronic regenerated state. A lack of responsiveness to serotonin appears to be the cause for the endothelial dysfunction.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.

Both Rho-kinase and the Ca(2+)/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca(2+). Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca(2+) and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein-coupled platelet receptors, independent of an increase in cytosolic Ca(2+). Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca(2+)/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).

Pertussis toxin is produced by the causative agent of whooping cough, Bordetella pertussis, and is an adenosine diphosphate (ADP)-ribosyltransferase capable of covalently modifying and thereby inactivating many eukaryotic G proteins involved in cellular metabolism. The toxin is a principal determinant of virulence in whooping cough and is a primary candidate for an acellular pertussis vaccine, yet it is unclear whether the ADP-ribosyltransferase activity is required for both pathogenic and immunoprotective activities. A B. pertussis strain that produced an assembled pertussis holotoxin with only 1 percent of the ADP-ribosyltransferase activity of the native toxin was constructed and was found to be deficient in pathogenic activities associated with B. pertussis including induction of leukocytosis, potentiation of anaphylaxis, and stimulation of histamine sensitivity. Moreover, this mutant strain failed to function as an adjuvant and was less effective in protecting mice from intracerebral challenge infection. These data suggest that the ADP-ribosyltransferase activity is necessary for both pathogenicity and optimum immunoprotection. These findings bear directly on the design of a nontoxic pertussis vaccine.

Seven mouse pigment mutants, which have alterations at distinct genes, are known to have a defect in kidney lysosomal enzyme secretion. Two of these, beige and pale ear, have a bleeding abnormality associated with a deficiency in the number of platelet dense granules. In the present study, five other mutants with defective lysosomal enzyme secretion--pearl, pallid, light ear, maroon, and ruby-eye--were likewise found to have abnormally prolonged bleeding times after experimental injury. Platelet counts were similar to those of normal mice, but the platelet dense granule components serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) and morphologically identifiable dense granules were markedly reduced in these mutants. The capacity to accumulate exogenous 3H-serotonin in platelets was reduced 2-3-fold. Thrombin-stimulated secretion of 3H-serotonin was slightly decreased in all mutants. However, the seven mutants could be subdivided into three groups based on the degree of secretion of lysosomal enzymes after thrombin stimulation. Thus, all seven mouse pigment mutants have symptoms consistent with platelet storage pool deficiency and may serve as useful animal models for specific types of human platelet storage pool disease. Also, the results emphasize the genetic, morphological, and functional interrelatedness of three organelles: melanosomes, lysosomes, and platelet dense granules.

Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.

Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF). The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation. Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation. The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.

OBJECTIVE

Heat shock protein (HSP) expression is vital to cellular and tissue protection after stress or injury. However, application of this powerful tool in human disease has been limited, as known enhancers of HSPs are toxic and not clinically relevant. Glutamine (GLN) can enhance HSP expression in non-clinically relevant animal injury models. The aim of this study was to assess the ability of GLN to enhance pulmonary HSP expression, attenuate lung injury, and improve survival after sepsis in the rat.

METHODS

Prospective, randomized, controlled animal trial.

METHODS

University research laboratory.

METHODS

Male Sprague-Dawley rats.

METHODS

We utilized a rat model of cecal ligation and puncture to induce sepsis. GLN or saline was administered 1 hr after initiation of sepsis via single tail-vein injection. We analyzed heat shock factor-1 phosphorylation, HSP-70, and HSP-25 via Western blot. Tissue metabolism was assayed by magnetic resonance spectroscopy. Occurrence of lung injury was determined via histopathologic examination. An inhibitor of HSP expression, quercetin, was utilized to assess role of HSP expression in prevention of sepsis-related mortality.

RESULTS

GLN, given after initiation of sepsis, enhanced pulmonary heat shock factor-1 phosphorylation, HSP-70, HSP-25, and attenuated lung injury after sepsis. Further, GLN improved indices of lung tissue metabolic function (adenosine 5-triphosphate/adenosine 5-diphosphate ratio, nicotinamide adenine dinucleotide) after sepsis. No significant effect of GLN on lung tissue-reduced glutathione was observed. GLN treatment led to a significant decrease in mortality (33% [6 of 18] GLN-treated rats vs. 78% [14 of 17] saline-treated rats). Administration of the HSP inhibitor quercetin blocked GLN-mediated enhancement of HSP expression and abrogated GLN's survival benefit.

CONCLUSIONS

GLN has been safely administered to critically ill patients and shown to improve outcome without clear understanding of the protective mechanism. Our results indicate GLN may prevent the occurrence of lung injury, lung tissue metabolic dysfunction, and mortality after sepsis via enhancement of deficient lung heat shock factor-1 phosphorylation/activation and HSP expression.

OBJECTIVE

This study assessed the effect of pharmacogenetics on the antiplatelet effect of clopidogrel.

BACKGROUND

Variability in clopidogrel response might be influenced by polymorphisms in genes coding for drug metabolism enzymes (cytochrome P450 [CYP] family), transport proteins (P-glycoprotein) and/or target proteins for the drug (adenosine diphosphate-receptor P2Y12).

METHODS

Sixty patients undergoing elective percutaneous coronary intervention in the randomized PRINC (Plavix Response in Coronary Intervention) trial had platelet function measured using the VerifyNow P2Y12 analyzer after a 600-mg or split 1,200-mg loading dose and after a 75- or 150-mg daily maintenance dosage. Polymerase chain reaction-based genotyping evaluated polymorphisms in the CYP2C19, CYP2C9, CYP3A4, CYP3A5, ABCB1, P2Y12, and CES genes.

RESULTS

CYP2C19*1*1 carriers had greater platelet inhibition 2 h after a 600-mg dose (median: 23%, range: 0% to 66%), compared with platelet inhibition in CYP2C19*2 or *4 carriers (10%, 0% to 56%, p = 0.029) and CYP2C19*17 carriers (9%, 0% to 98%, p = 0.026). CYP2C19*2 or *4 carriers had greater platelet inhibition with the higher loading dose than with the lower dose at 4 h (37%, 8% to 87% vs. 14%, 0% to 22%, p = 0.002) and responded better with the higher maintenance dose regimen (51%, 15% to 86% vs. 14%, 0% to 67%, p = 0.042).

CONCLUSIONS

Carriers of the CYP2C19*2 and *4 alleles showed reduced platelet inhibition after a clopidogrel 600-mg loading dose but responded to higher loading and maintenance dose regimens. Genotyping for the relevant gene polymorphisms may help to individualize and optimize clopidogrel treatment. (Australia New Zealand Clinical Trials Registry; ACTRN12606000129583).

Electrophoretic examination of 22-day-old, normal maize (Zea mays L.) endosperm extracts revealed two zones of adenosine diphosphate glucose pyrophosphorylase activity. The enzymes are identical in terms of Km for glucose 1-phosphate and the effect of 3-phosphoglyceric acid on apparent Km for glucose 1-phosphate. Both enzymatic activities increase with increasing doses of the functional alleles at the shrunken-2 and brittle-2 loci. Molecular weight differences between the two electrophoretic species were inferred from sucrose gradient centrifugation. It is suggested that the two bands of activity represent different aggregation states of the same enzyme because under different extraction conditions, only one enzyme is found. Molecular weight estimates of 237,000 and 253,000 were obtained for the smaller enzyme. It is suggested that this enzyme is an aggregate of several subunits. Comparison of the embryo and endosperm pyrophosphorylases showed the embryo activity to be more heat stable and probably independent of direct shrunken-2 or brittle-2 control.

BACKGROUND

The purpose of the present study was to study the concept of aspirin resistance or non-responsiveness by investigating the response to long-term aspirin therapy in patients with a former acute myocardial infarction (AMI).

METHODS

Patients with an AMI (n=202) randomly assigned to aspirin 160 mg/day (n=71), aspirin 75 mg/day and warfarin (INR 2.0-2.5) (n=58) or warfarin (INR 2.8-4.2) (n=73) were evaluated by the PFA-100(R), biochemical variables and clinical events after a mean treatment period of 4 years.

RESULTS

The limit for being an aspirin non-responder was defined as the 95th percentile value in the warfarin alone group (196 s) with the epinephrine cartridge. In patients on aspirin alone 25/71 (35%) were non-responders and on the combination 23/58 (40%). With the adenosine diphosphate (ADP) cartridge only minor differences were found. The levels of thromboxane B(2) in both aspirin groups, in responders as well as in non-responders, were extremely low compared to the warfarin alone group. Evaluating both aspirin groups together (n=129), the levels of soluble P-selectin were significantly higher in non-responders as compared to responders (p=0.012). During the observation period of 4 years with limited number of events, there was a tendency for higher event rates in non-responders as compared to responders (36% vs. 24%, p=0.28).

CONCLUSIONS

In our evaluation of the PFA-100(R) a considerable number of post-AMI patients seemed to be non-responders to long-term aspirin therapy in doses of 75 and 160 mg/day. Circulating levels of P-selectin were higher in the non-responders. A tendency to higher incidence of clinical events among non-responders was observed.

The interaction of cardiolipin with the isolated ADP/ATP carrier protein from beef heart mitochondria has been studied by means of the unmasking of a single cysteinyl residue, Cys56, which accompanies the conformational transition of the protein [Leblanc, P., & Clauser, H, (1972) FEBS Lett. 23, 107-113]. The unmasking was monitored by using the static fluorescence of the sulfhydryl reagent N-(1-pyrenyl)maleimide (PYM). The rate of PYM binding that was observed after initiation of the conformational transition by ADP was drastically reduced in the presence of cardiolipin (CL). Phospholipids other than CL were much less effective. It can be shown that the conformational transition and the binding reaction are both affected by CL, although to varying extents. An enhancement of the rate of the ADP-dependent PYM binding was observed upon digestion of the protein bound phospholipid by phospholipase A2. The phospholipase treatment also led to an increased ADP-independent PYM binding, thus indicating that the ADP control of the carrier transition was gradually lost. The ADP control could be fully restored through the addition of CL, provided that the phospholipase incubation had been terminated after approximately 1 h. These results will be discussed in relation to an earlier report of tight cardiolipin binding [Beyer, K., & Klingenberg, M. (1985) Biochemistry 24, 3821-3826] and to current structural models of the ADP/ATP carrier protein.

BACKGROUND

Cardioplegic arrest (CP) followed by reperfusion after cardiopulmonary bypass induces coronary microvascular dysfunction. We investigated the role of calcium-activated potassium (K(Ca)) channels in this dysfunction in the human coronary microvasculature.

RESULTS

Human atrial tissue was harvested before CP from a nonischemic segment and after CP from an atrial segment exposed to hyperkalemic cold blood CP (mean CP time, 58 minutes) followed by 10-minute reperfusion. In vitro relaxation responses of precontracted arterioles (80 to 180 mum in diameter) in a pressurized no-flow state were examined in the presence of K(Ca) channel activators/blockers and several other vasodilators. We also examined expression and localization of K(Ca) channel gene products in the coronary microvasculature using reverse transcriptase-polymerase chain reaction, immunoblot, and immunofluorescence photomicroscopy. Post-CP reperfusion relaxation responses to the activator of intermediate and small conductance K(Ca) channels (IK(Ca)/SK(Ca)), NS309 (10(-5) M), and to the endothelium-dependent vasodilators, substance P (10(-8) M) and adenosine 5diphosphate (10(-5) M), were significantly reduced compared with pre-CP responses (P<0.05, n=8/group). In contrast, relaxation responses to the activator of large conductance K(Ca) channels (BK(Ca)), NS1619 (10(-5) M), and to the endothelium-independent vasodilator, sodium nitroprusside (10(-4) M), were unchanged pre- and post-CP reperfusion (n=8/group). Endothelial denudation significantly diminished NS309-induced vasodilatation and abolished substance P- or adenosine 5 diphosphate-induced relaxation (P<0.05), but had no effect on relaxation induced by either NS1619 or sodium nitroprusside. The total polypeptide levels of BK(Ca), IK(Ca), and SK(Ca) and the expression of IK(Ca) mRNA were not altered post-CP reperfusion.

CONCLUSIONS

Cardioplegic arrest followed by reperfusion after cardiopulmonary bypass causes microvascular dysfunction associated with and likely in part due to impaired function of SK(Ca) and IK(Ca) channels in the coronary microcirculation. These results suggest novel mechanisms of endothelial and smooth muscle microvascular dysfunction after cardiac surgery.

Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)-mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate-ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.