Mito-FLAG with Ara-C as bolus versus continuous infusion in recurrent or refractory AML--long-term results of a prospective randomized intergroup study of the East German Study Group Hematology/Oncology (OSHO) and the Study Alliance Leukemia (SAL).

BACKGROUND: For patients with primary refractory or relapsed acute myeloid leukemia (AML), no treatment of choice has until now been defined to date. Cytarabine (Ara-C) is a key drug in the treatment of AML patients, there is still uncertainly regarding its optimal dose and infusion schedule. The aim of this study is to examine the impact of the Ara-C infusion schedule used as part of an intensive salvage regimen, in patients with relapsed or refractory AML. PATIENTS AND METHODS: A total of 252 adult patients (median age 59 years) with relapsed or refractory AML were randomly allocated to receive either Mito-FLAG with Ara-C as bolus (B) (1000 mg/m(2) over 1 h, every 12 h, days 1-5), or continuous infusion (CI) (150 mg/m(2) over 24 h, days 1-5) in combination with mitoxantrone, fludarabine, and granulocyte colony-stimulating factor (G-CSF). Autologous or allogeneic hematopoietic stem-cell transplantation was offered as consolidation therapy. Primary end point was the rate of complete remissions (CRs) after the first cycle of Mito-FLAG. RESULTS: The CR rates after Mito-FLAG (B) and Mito-FLAG (CI) were 54% and 43%, respectively (P = 0.1). There was no statistical difference between rates of grade 3/4 neutropenia, thrombocytopenia, mucositis, renal, and liver toxicity. More infections occurred, however, after Mito-FLAG (B) compared with Mito-FLAG (CI) (80% versus 69%, P = 0.01). The early death rate by day 42 was 13% in both arms. Median disease-free survival was comparable in the two arms (7.8 versus 7.1 months, P = 0.53) as was overall survival (7.1 versus 6.6 months, P = 0.53). CONCLUSION: A 5-day course of Ara-C 2 × 1000 mg/m(2) administered as bolus versus Ara-C 150 mg/m(2) administered by CI (in combination with mitoxantrone, fludarabine, and G-CSF), resulted in a nonsignificant trend in response rates in favor of Mito-FLAG (B) at the selected dose levels, but no differences in the survival outcome in relapsed or refractory AML. CLINICAL TRIAL NUMBER: LN_NN_2004_39/EudraCT number 2014-000083-18.

One-step RT-qPCR with an internal control system for the detection of turkey rotaviruses in faecal samples.

Turkey rotaviruses are one of the major pathogens responsible for the poult enteritis syndrome (PES). In this study a one step real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the rotaviral non-structural protein 4 (NSP4) was developed. The NSP4 is a highly conserved gene inside the turkey rotavirus genome and contains an internal control system to monitor any potential RT-qPCR inhibitors. The detection limit of the optimized NSP4-RT-qPCR assay ranged from 8.15 to 8.15 × 10(5) copy numbers. In total 149 faecal samples were collected from eight different flocks of commercial turkey farms. Faecal samples from hens and toms were collected separately at 2-week intervals from the 2nd week of age through the 16th and 20th week of age (age of slaughter for female and male, respectively) and tested. One farm reared only hens. The samples were tested previously using conventional RT-PCR targeting the same gene. When the conventional RT-PCR was compared with the developed NSP4-RT-qPCR, the results revealed that 11% of the samples of the conventional RT-PCR were false negative. The results indicate that this NSP4-RT-qPCR is highly sensitive for the detection of turkey rotaviruses in faeces. In addition, it could be suitable for the development of high-throughput screening.

Retrospective serological survey on selected viral pathogens in wild boar populations in Germany.

The objective of this study was to retrospectively evaluate the occurrence of porcine parvovirus (PPV), Aujeszky's disease virus (ADV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV) in selected wild boar populations in Germany (n = 1,221). Commercial enzyme linked immunosorbent assay and hemagglutination inhibition tests were used for serological monitoring. The serosurvey revealed seroprevalence rates of 64.28%, 11.26%, 7.87%, 7.84%, 3.82% and 1.59% for PPV, ADV, PRCV, SIV, PRRSV and TGEV, respectively. The seroprevalence rates differed between populations and age classes with the highest number of antibody-positive wild boars in older animals (>1 year old). No antibodies to TGEV were found in Baden-Wuerttemberg and in Mecklenburg-Western Pomerania (investigation period 1997/1998). In addition, sera collected in Mecklenburg-Western Pomerania in 1997/1998 were negative for SIV. Even though the seroprevalence rates established for these viruses, except for PPV, were relatively low, wild boars may act as a reservoir for pathogens and a source of infection for domestic pigs and humans. Based on the epidemiological situation, no risk of a spread of these viruses should emanate from wild boars, neither for wildlife nor for livestock. However, effective and science-based disease monitoring programmes should continuously be carried out in wild boar populations.

Diagnostic procedures after completion of oral immunisation against classical swine fever in wild boar.

The purpose of this paper is to define diagnostic procedures for wild boar after the completion of oral immunisation against classical swine fever (CSF). Epidemiological analysis of CSF in wild boar in Germany demonstrated that it is vital to carry out virological investigations on all animals found dead, sick or involved in traffic accidents. In principle, this should ensure an effective and prompt diagnosis of CSF. In addition, a defined number of wild boar, especially young animals < or = 6 months old, should also be tested for CSF virus to guarantee a high confidence level in the virological monitoring. Which animals should be examined serologically depends on the age class investigated, the season in which vaccination was stopped and the period of time since completion of vaccination. Therefore, different serological procedures have been defined for different situations during the first three years after completion of oral immunisation.

Activity of sirolimus in patients with myelodysplastic syndrome--results of a pilot study.

The pathophysiology of the myelodysplastic syndromes (MDS) involves disturbed regulation of angiogenesis, apoptosis, proliferation and differentiation as well as immune surveillance. Increasing data suggest that sirolimus might affect these pathways positively, thus being of possible therapeutic benefit in patients with this disease. Nineteen patients (n = 19) with a median age of 72 years (range 54-80 years) diagnosed with MDS received sirolimus orally with a target blood concentration of 3-12 ng/ml. Sirolimus was administered for a median of 3.7 months (range 0.3-11 months). Three patients [1 x refractory anaemia with excess blasts (RAEB)-2, 1 x RAEB-1, 1 x refractory cytopenia with multilineage dysplasia] showed either a major (1 x platelet, 1 x neutrophil) or a minor (1 x erythroid, 2 x platelet) haematological response according to International Working Group criteria. Major side-effects were hyperlipidaemia (n = 4), stomatitis (n = 3), thrombocytopenia (n = 2) and urinary tract infection (n = 1). These data suggest that sirolimus has activity in a subset of patients with more advanced MDS.

Substantial impact of FDG PET imaging on the therapy decision in patients with early-stage Hodgkin's lymphoma.

This prospective study assessed the impact of (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) on the staging and possible consequential changes of treatment regimen in patients with Hodgkin's lymphoma (HL). A total of 88 consecutive patients with histologically verified Hodgkin's lymphoma underwent a PET scan in addition to conventional staging procedures. Treatment was based on the conventional staging only, and the results of the FDG-PET did not affect the treatment strategy. The evaluation focused on the suggested change in clinical stage according to the Ann Arbor classification and on the suggested change in treatment strategy rather than on a lesion-by-lesion analysis. Using all the methods performed as the standard of reference, (18)F-FDG-PET staging was concordant with conventional staging in 70 out of 88 patients (80%). (18)F-fluorodeoxyglucose positron emission tomography suggested a change to a different clinical stage in 18 patients (20%). Management would have been changed in 16 patients (18%): intensification of treatment in nine patients (10%) and minimisation of treatment in seven patients (8%). In the 44 patients with early disease (stage IA-IIB), treatment would have been intensified in nine out of 44 patients (20%). (18)F-fluorodeoxyglucose positron emission tomography is a relevant noninvasive method that supplements conventional staging procedures and should therefore be used routinely to stage Hodgkin's lymphoma, particularly in patients with an early stage.

[Increased response rate with rituximab in relapsed and refractory follicular and mantle cell lymphomas -- results of a prospective randomized study of the German Low-Grade Lymphoma Study Group]. / Rituximab zur Remissionsinduktion bei rezidivierten und refraktären indolenten Lymphomen und Mantelzell-Lymphomen - Ergebnisse einer prospektiv randomisierten Studie der deutschen Studiengruppe für niedrig maligne Lymphome (GLSG) -

BACKGROUND AND OBJECTIVE: Rituximab has shown a high activity in relapsed follicular lymphomas when given alone. Further on, phase-II-studies indicate that its addition to chemotherapy may improve the response rate substantially. However, so far, prospective randomized studies have not been available. PATIENTS AND METHODS: In 1998 the GLSG started a multicenter trial in patients with relapsed or refractory indolent lymphoma or mantle cell lymphoma. A fludarabine-containing regimen (FCM) was chosen for salvage therapy, with fludarabine 25 mg/m(2)/d 1-3, cyclophosphamide 200 mg/m(2) d 1-3 and mitoxantrone 8 mg/m(2) d 1. A total of four courses, every 4 weeks were given. Patients were prospectively randomized for FCM alone or the immunochemotherapy with R-FCM (375 mg/m(2) one day before FCM) RESULTS: About 147 randomized patients 93 had follicular, 40 mantle cell and 14 lymphoplasmocytic/-cytoid lymphoma. Statistical analysis was performed by sequential testing and indicated for 94 fully evaluable patients a significant advantage for the R-FCM-arm, with an overall response rate of 83 % as compared to 58%, when treated with FCM alone (CR: 35 % vs. 13 %). Similar improvements of remission rate were detected in the different lymphoma subgroups, especially in MCL (OR: 65 % vs. 33 %). Both treatment options were associated with hematological toxicities of grade III and IV, but well tolerated; infectious complications were rare, with no difference between the two treatment groups. CONCLUSION: This prospectively randomized trial demonstrates for the first time a significant improvement of the combined immunochemotherapy related to the remission rate in patients with relapsed or refractory indolent lymphoma.

Busulfan, cyclophosphamide, and etoposide as high-dose conditioning regimen in patients with malignant lymphoma.

We investigated the efficacy and toxicity of the combination of busulfan, cyclophosphamide, and etoposide (Bu/Cy/VP-16) as a preparative regimen prior to autologous hematopoietic stem cell transplantation (ASCT) in patients with Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL). Fifty-three patients with recurrent ( n=30), refractory ( n=20), or high-risk ( n=3) lymphoma were enrolled. The 10 patients with HD and 43 with NHL (median age: 46 years, range: 18-64) received busulfan (16 mg/kg), cyclophosphamide (120 mg/kg), and etoposide (30 or 45 mg/kg) followed by ASCT. A total of 50 patients (94%) were consolidated in complete ( n=25) or partial ( n=25) remission, whereas 3 patients had chemoresistant disease before Bu/Cy/VP-16. Thirty-five patients (66%) had received prior radiotherapy (RT) excluding total body irradiation (TBI) as part of the conditioning regimen. The main nonhematological toxicities (grade II-IV according to the Bearman score) in 52 evaluable patients were mucositis (79%) and hepatic toxicity (15%). Severe veno-occlusive disease (VOD) occurred in three patients (5.8%) including one treatment-related death caused by VOD. Overall, treatment-related mortality was 3.8%. After a median follow-up for surviving patients of 21 months (range: 6-118), 20 patients (38%) are in continuous complete remission, 8 patients (15%) are alive in relapse, and 25 patients (47%) died. Probabilities of relapse, event-free survival, and overall survival at 3 years were 63% [95% confidence interval (CI): 48-79%], 31% (95% CI: 17-46%), and 43% (95% CI: 27-59%), respectively. In conclusion, Bu/Cy/VP-16 is an effective and well-tolerated conditioning regimen in patients with HD and NHL. Both toxicity and outcome were not significantly different in patients treated with 30 mg/kg and 45 mg/kg etoposide, respectively. The observed long-term results are even comparable to those published for other established high-dose protocols, including TBI-based regimens. However, further investigations are necessary to evaluate the value of Bu/Cy/VP-16 as a high-dose protocol for malignant lymphoma, especially in patients who have already received extensive RT.

Mito-flag as salvage therapy for relapsed and refractory acute myeloid leukemia.

BACKGROUND: This study was performed to examine the feasibility and toxicity of the combination of mitoxantrone, fludarabine, cytarabine as bolus (B) or continuous infusion (CI) and granulocyte- colony stimulating factor (G-CSF) in patients with recurrent and refractory acute myeloid leukemia (AML). PATIENTS AND METHODS: 29 patients with relapsed (n =17) or refractory (n =12) AML were treated with the Mito-FLAG protocol consisting of mitoxantrone (7 mg/m(2), days 1/3/5), fludarabine (15mg/m(2), every 12 h, days 1-5), cytarabine (Ara-C) as bolus infusion (1000 mg/m(2) over 1 h, every 12 h, days 1-5) (n =15) or as continuous infusion (100-150 mg/m(2) over 24 h, days 1-5) (n =14), and G-CSF (5 mgr;g/ kg/day, day 0 until a neutrophile count of 0.5 x10(9)/l). RESULTS: 17 patients (59%) and 1 patient (3%) achieved complete remission (CR) and partial remission (PR), respectively; thus the overall response rate was 62%. Following Mito-FLAG, 5 patients with CR underwent high-dose therapy (HDT) with allogeneic (n = 2) or autologous (n = 3) stem cell transplantation (SCT). With a median follow-up of 28 (range 6-54) months, 4 transplanted patients are alive in CR (n = 2) or in relapse (n = 2). The median duration of event-free survival (EFS) and overall survival (OS) was 3.2 and 6.8 months, and probabilities of EFS and OS after 1 year were 14 and 34%, respectively. The 1-year rates for EFS and OS in this group were 18 and 53%, respectively. Median duration of WHO grade 4 granulocytopenia and thrombocytopenia was 20 and 23 days, respectively. Nonhematological side effects were moderate, predominantly reaching WHO grades 1-2. Neutropenic fever was seen in 85% of courses, with a median duration of 4 (1-38) days. Four patients (14%) suffered an early death because of aplasia (n = 2), pneumonia (n =1) or progressive AML (1 nonresponding patient). CONCLUSIONS: Our data suggest that the Mito-FLAG protocol is feasible and can be safely performed with both schedules of Ara-C. In this study the regimens have shown high efficacy and acceptable toxicity in patients with relapsed or refractory AML. We currently examine the importance of bolus versus continuous infusion of Ara-C as part of the Mito-FLAG regimen in a prospective randomized multicenter trial.

Salvage chemotherapy with mitoxantrone, fludarabine, cytarabine, and cisplatin (MIFAP) in relapsing and refractory lymphoma.

PURPOSE: The aim of the study was to evaluate the feasibility and efficacy of the combination of mitoxantrone, fludarabine, cytarabine, and cisplatin (MIFAP) in patients with prognostically unfavorable recurrent and refractory Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). METHODS: Forty-six patients (median age 43 years, range 18-63) with relapsed (n = 15) or refractory (n = 31) malignant lymphoma were enrolled (HD, n = 13; low-grade/transformed NHL, n = 4; high-grade NHL, n = 29). A total of 39 patients (85%) showed multiply relapsed diseases with a duration of prior remission of < 12 months (n = 8) or had lymphoma being resistant to prior chemotherapy (n = 31). The MIFAP therapy consisted of fludarabine (15 mg/m2, q. 12 h, day 1-4), cytarabine (50 mg/m2 by continuous infusion (CI) over 22 h, day 1-4), cisplatin (25 or 30 mg/m2 by CI over 24 h, day 1-4), and mitoxantrone (4 mg/m2, day 2-5). RESULTS: Thirteen patients (28%) achieved complete remission (CR) and 15 patients (33%) partial remission (PR), for an overall response (OR) rate of 61%. Twenty-two patients responding to MIFAP (10 CR, 12 PR) have been consolidated by high-dose therapy (HDT) with hematopoietic stem cell transplantation (SCT). After a median follow-up of 12 months, 16 patients are in continuous CR (CCR) (n = 14) or CCRu (unconfirmed) (n = 2). The median duration of event-free survival (EFS) and overall survival (OS) were 6.5 and 19.3 months, respectively. Probabilities of EFS and OS after 3 years were 19% and 40%. Responders consolidated by subsequent HDT showed rates for 3-year EFS and OS of 40% and 66%, respectively. Unfavorable prognostic factors for EFS by univariate analysis were refractory lymphoma and the presence of B-symptoms. Significant prognostic factors for OS were NHL, refractory lymphoma, B-symptoms, and bone marrow involvement. The major toxicities were leukocytopenia and thrombocytopenia of the World Health Organization (WHO) grade IV in nearly all courses (median duration 10 and 11 days). In contrast, non-hematological side effects were moderate, predominantly of WHO grades I and II. Treatment-related mortality with MIFAP was 4% (two patients with septicemia by Aspergillus fumigatus). CONCLUSIONS: MIFAP is an effective salvage protocol for patients with poor-risk recurrent or refractory HD and NHL. The observed toxicity seems to be acceptable considering the unfavorable prognosis and intensive pretreatment. The results in patients responding to MIFAP and afterwards undergoing HDT with autologous stem cell support are even comparable to those published in patients with prognostically more favorable diseases.

ASHAP--an effective salvage therapy for recurrent and refractory malignant lymphomas.

BACKGROUND: This study was performed to examine the efficacy and toxicity of the combination of adriamycin (ADR), methylprednisolone (solumedrol), cytarabine (Ara-C), and cisplatin (CDDP) in patients with recurrent and refractory malignant lymphomas. PATIENTS AND METHODS: Sixty-five patients with Hodgkin's disease (HD) (n=14) or non-Hodgkin's lymphomas (NHL) (n = 51) were enrolled in the study. The ASHAP therapy consisted of ADR (40 mg/m2 by continuous infusion (CI) over 96 h), methylprednisolone (500 mg i.v., days 1-5), Ara-C (2 g/m2 as a 2-h infusion on day 5), and CDDP (100 mg/m2 by CI over 96 h). RESULTS: Twenty-five patients (38%) achieved complete remission (CR) and 20 (31%) were taken into partial remission (PR) for an overall response rate of 69%. Thirty-two patients with CR or PR following ASHAP underwent high-dose therapy (HDT) with subsequent hematopoietic stem cell transplantation. After a median follow-up of 52 months, 13 patients are in continuous CR (CCR), the 3-year event-free survival (EFS) was 30% for responders and 21% for all patients. The median overall survival (OS) was 12 months (range 0-70 months), and the OS rate after 3 years was 32%. Unfavorable prognostic factors for EFS and OS by univariate analysis were an elevated value of the serum lactate dehydrogenase and refractory lymphoma. The most frequently observed side effects following ASHAP were leukocytopenia and thrombocytopenia of World Health Organization (WHO) grades III/IV in approximately 80% of all courses. Non-hematological toxicities such as gastrointestinal side effects, infections, mucositis, renal and neurotoxicity occurred more rarely and reached WHO grades III/IV only occasionally. No treatment-related mortality with ASHAP was observed. CONCLUSIONS: ASHAP is an effective and moderately toxic salvage therapy for patients with recurrent or refractory HD and NHL. The results in patients responding to ASHAP and afterwards undergoing HDT with stem cell support are comparable with other established protocols and indicate an improvement in survival if HDT is carried out as intensification.

A reconstituted dilute blood clot lysis assay for the medium throughput screening of thrombolytic compounds.

Antithrombotic and clotting factors have long been targets for drug discovery, necessitating the development of blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted blood clot lysis assay which eliminates the need for on-site donors. The assay uses whole blood stored at 4 degrees C obtained from a local blood bank, diluted 1:10 in phosphate buffer. This blood was supplemented with 125I-labeled fibrinogen and the release of radioactive fibrinopeptides from formed clots was measured. The whole blood used in this assay, which had been stored at 4 degrees C for several days, no longer formed solid or retracting clots. Thus, platelets 5-7 days ex vivo (165 x 10(6) platelets) were added to the whole blood in the presence of thrombin (0.80 IU/ml) to form clots. Solid clots formed within 2 min of thrombin addition and began retracting shortly thereafter. In the absence of any thrombolytic agent, clots fully retracted within 2.5 h and remained stable. Thrombin-stimulated clot formation was completely inhibited by heparin. Clots could be lysed in a dose-dependent fashion in the presence of tissue-type plasminogen activator. Clot lysis could be completely inhibited in a dose-dependent fashion with plasminogen activator inhibitor type 1. To demonstrate the utility of this assay as a screen for thrombolytic agents, a 14-amino-acid PAI-1-inhibitory peptide relieved the PAI-1 effect on tPA in a dose-dependent fashion. These data describe an assay for the screening of potential pro-fibrinolytic agents that target PAI-1 inhibition in a human plasma-based system that is versatile, cost-effective, and physiologically relevant and does not rely on the availability of on-site blood donors.

Multiple enzymatic activities of the human cytosolic 85-kDa phospholipase A2: hydrolytic reactions and acyl transfer to glycerol.

The recombinant human 85-kDa cytosolic phospholipase A2 (cPLA2), when assayed in the presence of glycerol, catalyzes the transfer of acyl chains of radiolabeled phosphatidylcholine and para-substituted phenyl esters of fatty acids to glycerol, in addition to hydrolyzing these substrates. The product of the transacylation reaction is monoacylglycerol (MAG), and the acyl chain is predominantly esterified (> or = 95%) to a primary hydroxyl group of glycerol (sn-1/3); the stereochemistry is not known. Increasing concentrations of glycerol accelerate enzyme turnover both by providing an additional mechanistic pathway for the enzyme-substrate complex to form products and by increasing the intrinsic hydrolytic and transacylation activities of the enzyme. Significant enzymatic hydrolysis of sn-1/3-arachidonylmonoacylglycerol was measured, while sn-1/3-alpha-linolenoyl- and sn-2-arachidonylmonoacylglycerols were not detectably hydrolyzed. 1,3-Propanediol also serves as an acyl acceptor for the enzyme. cPLA2 hydrolyzes analog of lysophosphatidylcholine that lacks the sn-2 hydroxyl group. The enzyme will hydrolyze sn-1-acyl chains of rac-1-(arachidonyl, alpha-linolenoyl, palmitoyl)-2-O-hexadecyl-glycero-3-phosphocholine lipids and transfer the acyl chain to glycerol. Thus, cPLA2 has phospholipase A1 activity but only if an ether linkage rather than an ester linkage is present at the sn-2 position, and it is shown that the sn-1 acyl chains of both enantiomers of phosphatidylcholine are hydrolyzed. Phenyl [14C]-alpha-linolenate and five para-substituted phenyl esters of [3H]-alpha-linolenic acid with pKa values ranging from 7.2 to 10.2 for the phenol leaving groups were incorporated into 1,2-ditetradecyl-sn-glycero-3-phosphomethanol/Triton X-100 mixed micelles as substrates for the transacylation/hydrolysis reactions of the enzyme. Average product ratios, which are defined as the amount of monoacylglycerol formed to phenyl ester hydrolyzed, were 2.1 +/- 0.1 (n = 5) for the para-substituted phenyl esters and 2.0 +/- 0.3 (n = 7) for phenyl alpha-linolenate. The similarity of the ratios, despite the range of pKa values for the leaving groups, is consistent with the formation of a common enzyme intermediate that partitions to give either fatty acid or MAG. That intermediate may be a covalent acyl enzyme. Finally, the acyl chain specificity of cPLA2 was investigated to better understand the preference of the enzyme for phospholipids with sn-2-arachidonyl chains.

Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2.

Interfacial enzymes operate at an organized interface such as lipid aggregates in contact with the aqueous phase. The enzyme phospholipase A2 is a well studied interfacial enzyme, and a discussion of its behavior at interfaces is the topic of this review. Knowledge gained from studies of phospholipases A2 can be applied toward the quantitative analysis of other interfacial enzymes. The kinetic analysis of these enzymes is greatly simplified if one establishes certain experimental conditions that limit the exchange of enzyme and substrate between different substrate aggregates. With such constraints, the kinetics can be analyzed in terms of classical Michaelis-Menten theory adopted for the action of enzymes at interfaces. It is also possible to describe other enzyme properties such as inhibition and substrate preferences in a meaningful way using formalism that is well known in solution-phase enzymology.

Processive interfacial catalysis by mammalian 85-kilodalton phospholipase A2 enzymes on product-containing vesicles: application to the determination of substrate preferences.

Substrate specificities of the human and rat kidney 85-kDa phospholipase A2 enzymes (hmw-PLA2) have been determined under conditions in which hydrolysis of substrate vesicles occurs without the desorption of enzyme from the interface (scooting mode catalysis). The rat kidney enzyme binds to vesicles of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), which contain the substrate 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) and 10 mol% arachidonic acid (20:4) and 1-stearoyl-sn-glycero-3-phosphocholine (S-lyso-PC) as the hydrolysis reaction products, with a second-order rate constant k(on) approximately equal to 2 x 10(7) M-1 s-1. Upper limits of k(off) < or = 3 x 10(-4) s-1 and KD < = or 15 pM for the dissociation rate and equilibrium constants, respectively, are estimated from the vesicle binding measurements. The initial rates of hydrolysis of either radiolabeled 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphoserine (3H-SAPS), -phosphoethanolamine (3H-SAPE), -phosphoinositol (14C-SAPI), or -phosphate (3H-SAPA) and either 3H-SAPC or 14C-SAPC, which were incorporated into product-containing OPPC vesicles, were simultaneously measured with dual isotope radiometric assays. The plasmenylcholine 1-O-(Z-hexadec-1'-enyl)-2-arachidonyl-sn-glycero-3- phosphocholine (3H-PlasAPC) was also tested. Relative substrate specificity constants (Kcat/KM* values) were determined from the concentrations and initial rates of hydrolysis of the labeled substrates; the rank order of the values is SAPC approximately equal to SAPI approximately equal to PlasAPC > SAPE > SAPA approximately equal to SAPS. The maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discriminate between phospholipids with different polar head groups. The diglyceride 1-stearoyl-2-arachidonyl-sn-glycerol is not a substrate for the human hmw-PLA2. Two mixtures of 1-stearoyl-2-acyl-sn-glycero-3-phosphocholine, which have different sn-2 acyl chains, were prepared and compared to SAPC as substrates. One mixture contained naturally-occurring unsaturated fatty acyl chains and the other contained a mixture of 20:4, all of its partially hydrogenated analogues (20:3, 20:2, and 20:1), and arachidic acid (20:0). The order of preference for the human hmw-PLA2 is sn-2-20:4 > sn-2-alpha-linolenoyl > sn-2-linoleoyl > sn-2-oleoyl > or = sn-2-palmitoyleoyl.(ABSTRACT TRUNCATED AT 400 WORDS)

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change.

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).

Modification of anti-inflammatory drug effectiveness by ambient lipid peroxides.

When the steady-state concentrations of peroxide in prostaglandin H synthase assay systems was lowered by added glutathione peroxidase, several agents (meclofenamic acid, mefenamic acid, acetamidophenol and phenylbutazone) became more potent inhibitors of the prostaglandin-forming cyclooxygenase reaction. Paradoxically, these agents stimulated oxygen incorporation in the absence of added peroxidase. On the other hand, dithiothreitol, ibuprofen, flurbiprofen and indomethacin all inhibited the reaction in a dose-dependent manner, and their inhibitory potencies were unaffected by the action of glutathione peroxidase. Aspirin, dl-gamma-tocopherol and salicylic acid were not inhibitory (without preincubation) under the assay conditions employed in this study. The findings demonstrate that the potencies of some anti-inflammatory agents may be diminished by high local peroxide concentrations.

Phenolic anticyclooxygenase agents in antiinflammatory and analgesic therapy.

This report demonstrates that a non-steroidal antiinflammatory drug, MK 447, and phenol act similarly to inhibit cyclooxygenase activity in vitro in a dose-dependent manner when the concentration of peroxide activators is decreased by glutathione peroxidase. Increasing the rate of peroxide removal with higher amounts of glutathione peroxidase increases the inhibitory potency of the phenolic agents. The results support ascribing a vital role to tissue peroxides in facilitating prostaglandin biosynthesis in vivo. They also resolve the paradoxical problem of predicting antiinflammatory activity when an agent appears to stimulate prostaglandin formation. A corollary concept is that tissues with general hyperalgesic conditions may have lower levels of hydroperoxide than are found in inflammatory conditions, and thus be more amenable to therapy with phenolic agents like MK 447.

Dextran sulfates as a contaminant of DNA extracted from concentrated viruses and as an inhibitor of DNA polymerases.

Dextran sulfate is commonly used with polyethylene glycol to concentrate viruses before extraction of their DNA. However, dextran slulfate then easily contaminated such DNA and acted as a potent inhibitor of DNA polymerases from Bacillus subtilis (III), phage PBS2, and phage T4. Dextran sulfate only weakly inhibited Micrococcus luteus and Escherichia coli DNA polymerase I preparations.