Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Selective BH3-mimetics targeting BCL-2, BCL-XL or MCL-1 induce severe mitochondrial perturbations.

Induction of apoptosis by selective BH3-mimetics is currently investigated as a novel strategy for cancer treatment. Here, we report that selective BH3-mimetics induce apoptosis in a variety of hematological malignancies. Apoptosis is accompanied by severe mitochondrial toxicities upstream of caspase activation. Specifically, the selective BH3-mimetics ABT-199, A-1331852 and S63845, which target BCL-2, BCL-XL and MCL-1, respectively, induce comparable ultrastructural changes including mitochondrial swelling, a decrease of mitochondrial matrix density and severe loss of cristae structure. These shared effects on mitochondrial morphology indicate a similar function of these anti-apoptotic BCL-2 proteins in maintaining mitochondrial integrity and function.

Targeting intermediary metabolism enhances the efficacy of BH3 mimetic therapy in hematologic malignancies.

BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-XL (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.

A practical guide to graphic communication for quality assurance, education, and patient care in echocardiography.

Graphic communication (GC) is useful for continuous quality improvement (CQI), education, and patient care when in-person discussion is not possible because of geographic and schedule constraints. In echocardiography, these constraints can be mitigated by (a) capturing screenshots and device photos or videos and sharing them by email or text message, (b) simultaneous viewing of images on digital displays, and (c) broadcasting the study real time during acquisition to other mobile or stationary devices. Screenshots are useful for CQI and education and can be acquired, annotated, and shared with minimal impact on the flow of clinical echo interpretation. Providers at different locations can employ GC for shared clinical decision making by viewing echo studies from the same server, video conferencing or accessing real-time broadcast from a device. Which GC tool is selected is determined by its ease of use, the provider's goals and whether immediate image review is needed.

Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

BH3 profiling and a toolkit of BH3-mimetic drugs predict anti-apoptotic dependence of cancer cells.

BACKGROUND: Anti-apoptotic BCL-2 family members antagonise apoptosis by sequestering their pro-apoptotic counterparts. The balance between the different BCL-2 family members forms the basis of BH3 profiling, a peptide-based technique used to predict chemosensitivity of cancer cells. Recent identification of cell-permeable, selective inhibitors of BCL-2, BCL-XL and MCL-1, further facilitates the determination of the BCL-2 family dependency of cancer cells. METHODS: We use BH3 profiling in combination with cell death analyses using a chemical inhibitor toolkit to assess chemosensitivity of cancer cells. RESULTS: Both BH3 profiling and the inhibitor toolkit effectively predict chemosensitivity of cells addicted to a single anti-apoptotic protein but a combination of both techniques is more instructive when cell survival depends on more than one anti-apoptotic protein. CONCLUSIONS: The inhibitor toolkit provides a rapid, inexpensive and simple means to assess the chemosensitivity of tumour cells and in conjunction with BH3 profiling offers much potential in personalising cancer therapy.

Modulation of NADPH oxidase activity by known uraemic retention solutes.

BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. CONCLUSIONS: The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress.

Protein-bound uremic toxins stimulate crosstalk between leukocytes and vessel wall.

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

The enigma of fine mobile structures on the aortic surface in a patient undergoing transcatheter aortic valve replacement: a case report.

Background: The surface of the aorta generally does not show motion unless mobile atheroma, thrombi, vegetations, or intimal flaps are present. We previously described unusual mobile filamentous structures in the carotid artery. Here, we describe similar findings in the aorta and their possible cause. Case summary: An 88-year-old female with progressive exertional dyspnoea and severe aortic stenosis had a successful transcatheter aortic valve replacement (TAVR). A filamentous structure was noted on the focused pre-operative 2D transoesophageal echocardiography in the proximal descending aorta and post-TAVR as long strand-like structures attached to the thickened intimal wall with a planar component on 3D imaging. These findings were not associated with symptoms or clinical sequelae on short- and long-term follow-up. Discussion: The mobile structures that we describe are atypical for atheroma, thrombi, vegetations, and dissections in terms of their form and clinical presentation. 2D imaging showed that the filaments had focal thickening and emerged from the aortic surface. These findings suggest a relationship with the intima, perhaps from atherogenesis or injury with disruption or lifting of the intimal surface. No clinical sequelae were detected that may also relate to their position in the descending aorta and not the arch.

BCL2/BCL-X(L) inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Cinacalcet effect on polymorphonuclear leucocytes of kidney transplant patients.

BACKGROUND: Polymorphonuclear leucocytes (PMNLs) play a key role in the nonspecific immune defence. Cinacalcet reduces serum calcium levels in kidney transplant recipients with mineral bone disorder associated with chronic kidney disease. We investigated essential functions of PMNLs of kidney transplant recipients with and without hypercalcaemia and with and without cinacalcet therapy. SUBJECTS AND METHODS: Oxidative burst, phagocytosis, apoptosis and intracellular calcium concentrations of PMNLs from normocalcaemic kidney transplant patients without (KT-NC) or with cinacalcet intake (KT-NC/CI), hypercalcaemic kidney transplant patients (KT-HC) and healthy subjects (HS) were investigated. RESULTS: Stimulation of oxidative burst of PMNLs from KT-HC patients by phorbol-12-myristate-13-acetate or Escherichia coli was significantly attenuated compared with PMNLs from KT-NC, KT-NC/CI and HS. Apoptosis of PMNLs from KT-HC patients was significantly decreased compared with cells from KT-NC, KT-NC/CI and HS. Apoptosis correlated significantly with serum calcium concentrations. Intracellular calcium concentrations and phagocytosis of PMNLs did not differ between groups. CONCLUSIONS: Our data indicate that stimulation of PMNL oxidative burst and apoptosis is significantly diminished in kidney transplant patients with hypercalcaemia, while kidney transplant patients with serum calcium levels normalized by cinacalcet have normal PMNL functions despite immunosuppressive therapy.

The uraemic toxin phenylacetic acid contributes to inflammation by priming polymorphonuclear leucocytes.

BACKGROUND: The activation of polymorphonuclear leucocytes (PMNLs) causes inflammation and as a result cardiovascular disease, which is a main risk factor for increased morbidity and mortality in patients with chronic kidney disease. Toxins accumulating in uraemic patients play a major role in modulating essential PMNL functions and apoptosis, the latter being crucial for a coordinated resolution of inflammation. One uraemic toxin is phenylacetic acid (PAA). We therefore investigated whether PAA contributes to the deranged immune response in uraemia by modulating PMNL activities. METHODS: PMNL oxidative burst, phagocytosis and surface expression of the activation markers CD11b and CD18 were measured by flow cytometry in whole blood from healthy subjects in the presence and absence of PAA. Spontaneous apoptosis of isolated PMNLs was assessed by evaluating morphological features under the fluorescence microscope and by measuring the DNA content by flow cytometry. PMNL chemotaxis was tested by the under-agarose method. RESULTS: PAA significantly enhanced the stimulation of PMNL oxidative burst by Escherichia coli, phagocytosis of E. coli by PMNLs and the expression of CD11b and CD18 at the PMNL surface. PAA significantly decreased PMNL apoptosis resulting in an increased percentage of viable cells. PAA affected neither the oxidative burst stimulated by phorbol-12-myristate-13-acetate nor PMNL chemotaxis. CONCLUSIONS: PAA increases the activation of various PMNL functions and the expression of surface activation markers, while it attenuates PMNL apoptotic cell death. Therefore, PAA may contribute to the inflammatory state and consequently to increased cardiovascular risk in uraemic patients.

Race and epicardial fat: the impact of anthropometric measurements, percent body fat and sex.

OBJECTIVE: Epicardial fat is known to be thicker in White men than in Black men. The impact of sex, % body fat, and other anthropometric measures on epicardial fat thickness has not been described. Therefore we sought to evaluate how the racial differences in epicardial fat thickness would differ by these factors. METHODS: We used two-dimensional transthoracic echocardiography to measure the epicardial fat thickness in 150 patients who were admitted to our clinical decision unit for chest pain. Standard anthropometric measurements were performed and body mass index (BMI) and % body fat were calculated. Data were analyzed using analysis of variance and multiple regression. RESULTS: Epicardial fat measured at the mid right ventricular wall was significantly greater in Whites than Blacks (4.9 +/- 2.1 mm vs 3.8 +/- 1.8 mm, for males, and 5.8 +/- 3.2 mm vs 3.7 +/- 1.7 mm, for females). The results from regression analysis showed that after controlling for age, sex, BMI and waist circumference, race remained a significant predictor of epicardial fat, with Whites having higher amounts of fat than Blacks. The difference by race remained even after controlling for % body fat, which was also a significant predictor. CONCLUSION: Anterior epicardial fat thickness is greater in White than Black men and women of the same race and is independent of anthropometric measurements and % body fat. Race may be an important consideration when analyzing the relationship between epicardial fat and cardiovascular risk.

Normal and pathologic concentrations of uremic toxins.

An updated review of the existing knowledge regarding uremic toxins facilitates the design of experimental studies. We performed a literature search and found 621 articles about uremic toxicity published after a 2003 review of this topic. Eighty-seven records provided serum or blood measurements of one or more solutes in patients with CKD. These records described 32 previously known uremic toxins and 56 newly reported solutes. The articles most frequently reported concentrations of ß2-microglobulin, indoxyl sulfate, homocysteine, uric acid, and parathyroid hormone. We found most solutes (59%) in only one report. Compared with previous results, more recent articles reported higher uremic concentrations of many solutes, including carboxymethyllysine, cystatin C, and parathyroid hormone. However, five solutes had uremic concentrations less than 10% of the originally reported values. Furthermore, the uremic concentrations of four solutes did not exceed their respective normal concentrations, although they had been previously described as uremic retention solutes. In summary, this review extends the classification of uremic retention solutes and their normal and uremic concentrations, and it should aid the design of experiments to study the biologic effects of these solutes in CKD.

Effect of Hydrogen Sulfide on Essential Functions of Polymorphonuclear Leukocytes.

Impaired polymorphonuclear leukocyte (PMNL) functions contribute to increased infections and cardiovascular diseases in chronic kidney disease (CKD). Uremic toxins reduce hydrogen sulfide (H2S) levels and the anti-oxidant and anti-inflammatory effects of H2S. Its biosynthesis occurs as a side process of transsulfuration and in the disposal of adenosylhomocysteine, a transmethylation inhibitor and proposed uremic toxin. PMNL chemotaxis was measured by the under-agarose method, phagocytosis, and oxidative burst by flow cytometry in whole blood and apoptosis by determining DNA content by flow cytometry and morphological features by fluorescence microscopy. Sodium hydrogen sulfide (NaHS), diallyl trisulphide (DATS) and diallyl disulphide (DADS), cysteine, and GYY4137 were used as H2S-producing substances. Increased H2S concentrations did not affect chemotaxis and phagocytosis. NaHS primed PMNL oxidative burst activated by phorbol 12-myristate 13-acetate (PMA) or E. coli. Both DATS and cysteine significantly decreased E. coli-activated oxidative burst but had no effect on PMA stimulation. While NaHS, DADS, and cysteine attenuated PMNL apoptosis, GYY4137 decreased their viability. Experiments with signal transduction inhibitors suggest that the intrinsic apoptosis pathway is mainly involved in GYY4137-induced PMNL apoptosis and that GYY4137 and cysteine target signaling downstream of phosphoinositide 3-kinase.

Determination of insoluble, soluble, and total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: collaborative study.

A method for the determination of insoluble (IDF), soluble (SDF), and total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official Methods 985.29, 991.43, 2001.03, and 2002.02, the method quantitates water-insoluble and water-soluble dietary fiber. This method extends the capabilities of the previously adopted AOAC Official Method 2009.01, Total Dietary Fiber in Foods, Enzymatic-Gravimetric-Liquid Chromatographic Method, applicable to plant material, foods, and food ingredients consistent with CODEX Definition 2009, including naturally occurring, isolated, modified, and synthetic polymers meeting that definition. The method was evaluated through an AOAC/AACC collaborative study. Twenty-two laboratories participated, with 19 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 10.45 to 29.90%. Digestion of samples under the conditions of AOAC 2002.02 followed by the isolation, fractionation, and gravimetric procedures of AOAC 985.29 (and its extensions 991.42 and 993.19) and 991.43 results in quantitation of IDF and soluble dietary fiber that precipitates (SDFP). The filtrate from the quantitation of water-alcohol-insoluble dietary fiber is concentrated, deionized, concentrated again, and analyzed by LC to determine the SDF that remains soluble (SDFS), i.e., all dietary fiber polymers of degree of polymerization = 3 and higher, consisting primarily, but not exclusively, of oligosaccharides. SDF is calculated as the sum of SDFP and SDFS. TDF is calculated as the sum of IDF and SDF. The within-laboratory variability, repeatability SD (Sr), for IDF ranged from 0.13 to 0.71, and the between-laboratory variability, reproducibility SD (SR), for IDF ranged from 0.42 to 2.24. The within-laboratory variability Sr for SDF ranged from 0.28 to 1.03, and the between-laboratory variability SR for SDF ranged from 0.85 to 1.66. The within-laboratory variability Sr for TDF ranged from 0.47 to 1.41, and the between-laboratory variability SR for TDF ranged from 0.95 to 3.14. This is comparable to other official and approved dietary fiber methods, and the method is recommended for adoption as Official First Action.

Percutaneous thrombectomy and right ventricular mechanical circulatory support for pulmonary embolism in a coronavirus disease 2019 patient: case report, 1-year update, and echocardiographic findings.

BACKGROUND: We previously described percutaneous thrombectomy and right ventricular (RV) mechanical support of a coronavirus disease 2019 (COVID-19) patient with a massive pulmonary embolism. Here, we present a detailed echocardiographic and clinical timeline with 1-year follow-up. CASE SUMMARY: A 57-year-old female with COVID-19 went into shock from a massive pulmonary embolism. After percutaneous removal of a large thrombus burden (AngioVac system; AngioDynamics Inc., Latham, NY, USA), she became severely hypotensive, requiring cardiopulmonary resuscitation, and hemodynamic support with an Impella RP device (Abiomed, Danvers, MA, USA). A paediatric transoesophageal echocardiography (TOE) probe monitored the procedure because an adult probe would not pass (S7-3t-Philips Medical Systems, Andover, MA, USA). Post-thrombectomy, surface imaging documented gradual resolution of RV dysfunction, tricuspid regurgitation, and elevated pulmonary artery pressure. Her course was complicated by renal failure requiring temporary dialysis. She was discharged home on apixaban. Hypercoagulability work-up was negative. Two months later, vocal cord surgery was performed for persistent stridor. Esophagoscopy at that time was prevented by osteophyte obstruction. At 10 months, she received the Pfizer-BioNTech vaccine. At 1 year, the patient remains healthy on apixaban, and her echocardiogram is normal. DISCUSSION: This case illustrates the pivotal role of echocardiography in the diagnosis, percutaneous treatment, and near- and long-term follow-up and management of a patient with massive pulmonary embolism due to COVID-19 with documentation of complete recovery from severe RV dysfunction and haemodynamic collapse. A paediatric TOE probe was a crucial alternative to the adult probe because of possible osteophyte obstruction.

Novel roles of RTN4 and CLIMP-63 in regulating mitochondrial structure, bioenergetics and apoptosis.

The recruitment of DRP1 to mitochondrial membranes prior to fission is facilitated by the wrapping of endoplasmic reticulum (ER) membranes around the mitochondria. To investigate the complex interplay between the ER membranes and DRP1 in the context of mitochondrial structure and function, we downregulate two key ER shaping proteins, RTN4 and CLIMP-63, and demonstrate pronounced mitochondrial hyperfusion and reduced ER-mitochondria contacts, despite their differential regulation of ER architecture. Although mitochondrial recruitment of DRP1 is unaltered in cells lacking RTN4 or CLIMP-63, several aspects of mitochondrial function, such as mtDNA-encoded translation, respiratory capacity and apoptosis are significantly hampered. Further mechanistic studies reveal that CLIMP-63 is required for cristae remodeling (OPA1 proteolysis) and DRP1-mediated mitochondrial fission, whereas both RTN4 and CLIMP-63 regulate the recruitment of BAX to ER and mitochondrial membranes to enable cytochrome c release and apoptosis, thereby performing novel and distinct roles in the regulation of mitochondrial structure and function.

Implications of the inability of Listeria monocytogenes EGD-e to grow anaerobically due to a deletion in the class III NrdD ribonucleotide reductase for its use as a model laboratory strain.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that causes life-threatening diseases in humans. It grows and survives in environments of low oxygen tension and under conditions of strict anaerobiosis. Oxygen-limiting conditions may be an important factor in determining its pathogenicity. L. monocytogenes serovar 1/2a strain EGD-e has been employed intensively to elucidate the mechanisms of intracellular multiplication and virulence. Listeria possesses genes encoding class I aerobic and class III anaerobic ribonucleotide reductases (RNRs). The class III RNR consists of a catalytic subunit NrdD and an activase NrdG. Surprisingly, L. monocytogenes EGD-e, but not other L. monocytogenes strains or other listerial species, is unable to grow under strict anaerobic conditions. Inspection of listerial NrdD amino acid sequences revealed a six-amino acid deletion in the C-terminal portion of the EGD-e protein, next to the essential glycyl radical domain. Nevertheless, L. monocytogenes EGD-e can grow under microaerophilic conditions due to the recruitment of residual class Ia RNR activity. A three-dimensional (3D) model based on the structure of bacteriophage T4 NrdD identified the location of the deletion, which appears in a highly conserved part of the NrdD RNR structure, in the α/ß barrel domain near the glycyl radical domain. The deleted KITPFE region is essential either for interactions with the NrdG activase or, indirectly, for the stability of the glycyl radical loop. Given that L. monocytogenes EGD-e lacks a functional anaerobic RNR, the present findings are relevant to the interpretation of studies of pathogenesis with this strain specifically, in particular under conditions of low oxygen tension.

The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein.

Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using (3)H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.