Lysate Assay Research Articles

From pH to MALDI-TOF: Hundreds of spotted opportunities?

Current protocols for quality assurance of platelet concentrates used in transfusion therapy include evaluation of platelet count and pH, in vitro measurements of platelet lysis, membrane activation and microparticle release and assays of platelet ability to respond to aggregation stimuli and to hypotonic shock. Unfortunately, these assays show limited correlation to post-transfusion platelet survival and recovery in the recipient. This requires validation of platelet collection and storage systems with expensive and time consuming autologous transfusion studies in healthy volunteers with radiolabeled platelets. Furthermore, platelets from some donors show increased lesion during storage for reasons that are incompletely understood. This editorial discusses recent strides in proteomic technology which open interesting perspectives for improving current procedures for quality assurance of platelet concentrates and increasing the safety and effectiveness of platelet transfusion in medical and surgical conditions. This article is part of a Special Issue entitled: Integrated omics.

Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC50s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1pgcell−1 for 13-desmethyl spirolide C, 96.4pgcell−1 for 12-methylgymnodimine and a potent hemolytic EC50 value of 7.1×103 cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.

A hypercoagulable and hypofibrinolytic state is detectable by global methods in patients with retinal vein occlusion

The pathogenesis of retinal vein occlusion (RVO), has not been well understood. Recent data have shown the efficacy of an anticoagulant therapy with LMWHs in the treatment of acute RVO suggesting the presence of a hypercoagulable state in these patients. New global tests for detection of hypercoagulability and hypofibrinolysis have become available and their application might improve the knowledge of the pathophysiology of RVO and, potentially, its treatment. The aim of our study was to evaluate coagulation and fibrinolytic alterations by two global tests in RVO patients: Endogenous Thrombin Potential (ETP) and Clot Lysis Time (CLT), respectively. We studied 81 RVO patients (40 males; median age 61 years) and a control group matched for age and sex. The ETP was measured by functional chromogenic assay and expressed as the time until thrombin burst (LagTime), Time to peak (Tmax), Peak amount of thrombin generation (Cmax) and ETP. CLT was determined by a plasma-based, tissue factor-induced clot lysis assay. Cmax, ETP and CLT values were significantly higher in RVO patients than in controls (Cmaxp = 0.010; ETP p < 0.001; CLT p < 0.001) and remained significantly associated with the disease at the multivariate analysis adjusted for cardiovascular risk factors. Our results indicate that -beyond the assay of different parameters associated with clotting activation and lysis- global methods might allow us to easily detect the presence of hypercoagulability and hypofibrinolysis in RVO patients. Further studies should assess the possible clinical value of our data in the management of RVO patients.

Intact thrombin generation and decreased fibrinolytic capacity in patients with acute liver injury or acute liver failure

It has been well established that hemostatic potential in patients with chronic liver disease is in a rebalanced status due to a concomitant decrease in pro- and antihemostatic drivers. The hemostatic changes in patients with acute liver injury/failure (ALI/ALF) are similar but not identical to the changes in patients with chronic liver disease and have not been studied in great detail. To assess thrombin generation and fibrinolytic potential in patients with ALI/ALF. We performed thrombin generation tests and clot lysis assays in platelet-poor plasma from 50 patients with ALI/ALF. Results were compared with values obtained in plasma from 40 healthy volunteers. The thrombin generation capacity of plasma from patients with ALI/ALF sampled on the day of admission to hospital was indistinguishable from that of healthy controls, provided thrombomodulin was added to the test mixture. Fibrinolytic capacity was profoundly impaired in patients with ALI/ALF on admission (no lysis in 73.5% of patients, compared with 2.5% of the healthy controls), which was associated with decreased levels of the plasminogen and increased levels of plasminogen activator inhibitor type 1. The intact thrombin generating capacity and the hypofibrinolytic status persisted during the first week of admission. Patients with ALI/ALF have a normal thrombin generating capacity and a decreased capacity to remove fibrin clots. These results contrast with routine laboratory tests such as the PT/INR, which are by definition prolonged in patients with ALI/ALF and suggest a bleeding tendency.

Development of a high throughput assay for indirectly measuring phage growth using the OmniLogTMsystem

The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. Here we describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. Here we show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, we show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.

Toxicity profile of commercially produced indigenous banana beer

Mycotoxins, together with endotoxins, represent important classes of naturally occurring contaminants in food products, posing significant health risks to consumers. The aim of this study is to investigate the occurrence of both Fusarium mycotoxins and endotoxins in commercially produced traditional banana beer. Two brands of commercially produced traditional banana beer were collected from a local retail market in Kigali, Rwanda. Beer samples were analysed for the presence of deoxynivalenol (DON), fumonisin B1 and zearalenone (ZEA), using an enzyme-linked immuno-sorbent assay (ELISA) method. The quantification of bacterial endotoxin using Limulus amoeboecyte lysate (LAL) assay was also conducted. The contamination levels were 20 and 6.7 µg kg−1 for DON; 34 and 31.3 µg kg−1 for FB1; 0.66 and 2.2 µg kg−1 for ZEA in brands A and B of the beers, respectively. Results indicate that the levels of Fusarium toxins and bacterial endotoxin reported in this study did not pose adverse human health effects as a result of drinking/consuming banana beer. However, exposure to low/sub-threshold doses or non-toxic levels of endotoxins magnifies the toxic effect of xenobiotic agents (e.g. fungal toxins) on liver and other target organs. Considering Fusarium toxins and/or endotoxin contamination levels in other agricultural commodities intended for human consumption, health risks might be high and the condition is aggravated when beer is contaminated by mixtures of the mycotoxins, as indicated in this study.

New Sequence-Defined Polyaminoamides with Tailored Endosomolytic Properties for Plasmid DNA Delivery

Heterogeneity of polymeric carriers is one of the most elusive obstacles in the development of nonviral gene delivery systems, concealing interaction mechanisms and limiting the use of structure-activity relationship studies. In this report, novel sequence-defined polyaminoamides, prepared by solid-phase assisted synthesis, were used to establish first structure-activity relationships for polymer-based plasmid DNA delivery. By combining a cationic building block with hydrophobic modifications and bioreversible disulfide cross-linking sites, transfection polymers with tailored lytic and DNA binding properties were designed. These polymers demonstrated clear correlation between structure and performance in lysis and DNA binding assays. In vitro studies showed negligible toxicity and highly efficient gene transfer, demonstrating the potential of this platform in the fast, combinatorial development of new transfection polymers.

An atomic-force basis for the bacteriolytic effects of granulysin

While granulysin has been suggested to play an important role in adaptive immune responses against bacterial infections by killing pathogens, and molecular force for protein–protein interaction or protein–bacteria interaction may designate the specific functions of a protein, the molecular-force basis underlying the bacteriolytic effects of granulysin at single-molecule level remains unknown. Here, we produced and purified bactericidal domain of macaque granulysin (GNL). Our bacterial lysis assays suggested that GNL could efficiently kill bacteria such as Listeria monocytogenes. Furthermore, we found that the interaction force between GNL and L. monocytogenes measured by an atomic force microscopy (AFM) was about 22.5pN. Importantly, our AFM-based single molecular analysis suggested that granulysin might lyse the bacteria not only through electrostatic interactions but also by hydrogen bonding and van der Waals interaction. Thus, this work provides a previous unknown mechanism for bacteriolytic effects of granulysin.

Oxidized Phospholipids Are Present on Plasminogen, Affect Fibrinolysis, and Increase Following Acute Myocardial Infarction

This study sought to assess whether plasminogen, which is homologous to lipoprotein (a) [Lp(a)], contains proinflammatory oxidized phospholipids (OxPL) and whether this has clinical relevance. OxPL measured on apolipoprotein B-100 (OxPL/apoB), primarily reflecting OxPL on Lp(a), independently predict cardiovascular disease (CVD) events. The authors examined plasminogen from commercially available preparations and plasma from chimpanzees; gorillas; bonobos; cynomolgus monkeys; wild-type, apoE(-/-), LDLR(-/-), and Lp(a)-transgenic mice; healthy humans; and patients with familial hypercholesterolemia, stable CVD, and acute myocardial infarction (AMI). Phosphocholine (PC)-containing OxPL (OxPC) present on plasminogen were detected directly with liquid chromatography-mass spectrometry (LC-MS/MS) and immunologically with monoclonal antibody E06. In vitro clot lysis assays were performed to assess the effect of the OxPL on plasminogen on fibrinolysis. LC-MS/MS revealed that OxPC fragments were covalently bound to mouse plasminogen. Immunoblot, immunoprecipitation, density gradient ultracentrifugation, and enzyme-linked immunosorbent assay analyses demonstrated that all human and animal plasma samples tested contained OxPL covalently bound to plasminogen. In plasma samples subjected to density gradient fractionation, OxPL were present on plasminogen (OxPL/plasminogen) in non-lipoprotein fractions but on Lp(a) in lipoprotein fractions. Plasma levels of OxPL/apoB and OxPL/apo(a) varied significantly (>25×) among subjects and also strongly correlated with Lp(a) levels. In contrast, OxPL/plasminogen levels were distributed across a relatively narrow range and did not correlate with Lp(a). Enzymatic removal of OxPL from plasminogen resulted in a longer lysis time for fibrin clots (16.25 vs. 11.96 min, p = 0.007). In serial measurements over 7 months, OxPL/plasminogen levels did not vary in normal subjects or in patients with stable CVD, but increased acutely over the first month and then slowly decreased to baseline in patients following AMI. These data demonstrate that plasminogen contains covalently bound OxPL that influence fibrinolysis. OxPL/plasminogen represent a second major plasma pool of OxPL, in addition to those present on Lp(a). OxPL present on plasminogen may have pathophysiological implications in AMI and atherothrombosis.

Alexandrium peruvianum (Balech and Mendiola) Balech and Tangen a new toxic species for coastal North Carolina

Routine sampling of the water quality stations in the New River Estuary (Jacksonville, North Carolina, USA) during November 2004 revealed the presence of a previously unidentified dinoflagellate. Preliminary observations of its morphology suggested it to be consistent with that of Alexandrium peruvianum (Balech et Mendiola) Balech et Tangen. Observations using brightfield, epifluorescence and scanning electron microscopy confirmed the diagnostic thecal plates to be those of A. peruvanium. Clonal cultures established from cells isolated from the New River Estuary samples were also used for further studies of morphology and for the presence of toxins. Thecal morphology was consistent with that described by Balech clearly separating it from the sister species Alexandrium ostenfeldii. Three classes of toxins were detected from these cultures. An erythrocyte lysis assay (ELA) was used to confirm the presence of hemolytic toxins in A. peruvianum cultures. A cellular EC50 for lysis was 1.418×104 cells, well within the range the maximal cells densities found in the New River and more potent when compared on a cellular basis with Prymnesium parvum. Another toxin class detected in A. peruvianum cultures was the fast acting 13-desmethy C and D spirolides also produced by the sister species A. ostenfeldii. The last toxin type detected in the A. peruvianum cultures was the paralytic shellfish toxins, GTX 2, 3, B1, STX and C1,2. These findings expand the geographic range of occurrence for A. peruvianum in the U.S. to be much greater than previously considered. The morphological characters agreed with previously reported molecular data in separating A. peruvianum from A. ostenfeldii. It is also the first confirmed report that this species produces PSP toxins, spirolides and naturally occurring hemolytic substances. In light of these findings additional attention is needed for the detection of Alexandrium species in all coastal waters of the U.S. This added effort will enhance the evaluation of the relative impacts of the species to shellfish safety and bloom surveillance.

Reduced clot-stability during the first 6 hours after aneurysmal subarachnoid haemorrhage – a prospective case-control study

IntroductionEarly rebleeding is an important cause of death and disability following aneurysmal subarachnoid haemorrhage (SAH). Recent studies have shown that 50-90% of the rebleedings occurred within the first 6hours after the primary bleeding. The mechanism leading to rebleeding remains to be established. In the present prospective case-control study we hypothesize that patients with SAH develop a coagulopathy characterized by reduced clot stability during the early period after the initial bleeding. MethodsPatients with aneurysmal SAH was studied with a dynamic clot lysis assay and markers of fibrinolysis and clot stabilizers in blood samples taken within and after 6hours after onset of bleeding. Results were compared with blood samples from age and gender matched healthy controls. Results36 patients were enrolled, 26 patients had blood samples collected within 6hours after the initial bleeding whereas 10 patients had blood samples taken later than 6hours after the initial bleeding. Patients demonstrated significantly reduced clot stability during the first 6hours after initial bleeding. Fibrinolytic activity was increased during the first 6hours along with the inhibitors of fibrinolysis whereas the modulators of fibrinolysis were reduced or inactivated. ConclusionDuring the first 6hours after SAH patients exhibit reduced clot-stability. Probably a consequence of activated fibrinolysis in combination with reduced or inactivated factor XIII and thrombin-activable fibrinolysis inhibitor.

Combinatory effects of hepatic CD8+ and NK lymphocytes in bile duct injury from biliary atresia

To our knowledge, elucidating the immune pathogenesis of disease, especially characteristic T-cell and natural killer (NK) cell expansions, has not been performed before now. We investigated the role of T lymphocytes and NK lymphocytes in the destruction of extrahepatic bile ducts of patients with biliary atresia. Lymphocytes from the liver and extrahepatic bile duct remnants of patients with biliary atresia were characterized by immunofluorescence staining, fluorescence-activated cell sorter analysis, and real-time reverse-transcriptase PCR. Cholangiocyte lysis assays were performed to confirm cytotoxicity of activated hepatic NK lymphocytes or CD8(+) cells. The inflammatory milieu from portal tracts and/or biliary remnants consisted of greater numbers of Kupffer cells, T lymphocytes, and NK lymphocytes in the patients with biliary atresia as compared with the cholestatic and noncholestatic controls. In patients with biliary atresia, expression of NK or CD8+ costimulatory molecules was upregulated as compared with controls. Hepatic NK lymphocytes or CD8(+) cells from patients with biliary atresia were demonstrated to be cytotoxic to the duct epithelium. Specific immune responses from NK and CD8(+) cells were involved in the injury to the duct epithelium and play a significant role in the phenotype of experimental biliary atresia.

Utility of Clot Formation and Lysis Assay to Monitor Global Coagulation State of Patients with Severe Acute Pancreatitis

Systemic inflammation affects hemostasis during severe acute pancreatitis (SAP). A hypercoagulable state occurs more frequently in SAP, which is not fully detected by traditional coagulation testing. The aim of this study was to evaluate the contribution of clot formation and lysis (CloFAL) assay to improve monitoring of global coagulation in patients with SAP. Twenty-five patients with SAP who were treated from December 2009 to April 2011 were studied. Plasma was collected at the time of admission, and CloFAL was measured using the CloFAL analyzer. The parameters evaluated include coagulation time (CT), fibrinolysis time (FT), and maximum amplitude (MA), from which the accelerating coagulation extent (ACE, MA/CT), accelerating fibrinolytic extent (AFE, MA/FT), and balance level exponent (BLE, ACE/AFE) were calculated. In addition, laboratory values for the traditional coagulation testing were measured. Values were compared to a control group of 20 healthy subjects. The MA, FT, ACE, and BLE values of the CloFAL assay were significantly increased in the SAP group compared to the control group (p\0.05 for all measurements). For the traditional coagulation testing, fibrinogen, plasminogen, and D-dimer levels were higher in patients in the SAP group compared to the control group (p\0.05). Our findings using the CloFAL analyzer indicate that the hypercoagulable state was due to increased fibrin generation and invariable fibrinolysis in patients with SAP. CloFAL assay is a simple and useful global coagulation assay to monitor hypercoagulable states during SAP.

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.

Complement C3 is a novel plasma clot component with anti-fibrinolytic properties

Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes. Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components. Analysis of paired plasma and serum samples confirmed concentration-dependent incorporation of C3 into clots. Surface plasmon resonance indicated high-affinity binding interactions between C3 and fibrinogen and fibrin. Turbidimetric clotting and lysis assays indicated C3 impaired fibrinolysis in a concentration-dependent manner, both in vitro and ex vivo. These data indicate functional interactions between complement C3 and fibrin leading to prolonged fibrinolysis. These interactions are physiologically relevant in the context of protection following injury and suggest a mechanistic link between increased plasma C3 concentration and acute cardiovascular thrombotic events.

Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay.

DETERMINATION OF STREPTOKINASE ACTIVITY IS USUALLY ACCOMPLISHED THROUGH TWO ASSAY METHODS: a) Clot lysis, b) Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase®, which is a native product and Heberkinasa®, which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase® was found to be 101 ± 4% and 97 ± 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa® the potency values obtained were 42 ± 5% and 92.5 ± 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein.

Evidence that fibrinolytic changes in paediatric obesity translate into a hypofibrinolytic state

Paediatric obesity, like adulthood obesity, is associated with an increase of fibrinolysis inhibitors. No study, however, has evaluated the impact of these changes on plasma fibrinolytic capacity. We investigated plasma fibrinolysis and the role therein of the fibrinolytic changes associated with obesity in 59 obese children (body mass index > 95th percentile) and 40 matched controls. Fibrinolysis was investigated by measuring 1) the plasma levels of relevant fibrinolytic factors; 2) the in vitro fibrinolytic capacity under different conditions, using a microplate plasma clot lysis assay; 3) the circulating levels of markers of clotting and fibrinolysis activation. Plasminogen activator inhibitor 1 (PAI-1), total thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen levels were higher in obese children as compared to controls (p<0.01). Plasma clots from obese children lysed significantly slower than control clots when exposed to exogenous plasminogen activator, indicating a greater resistance to fibrinolysis. By the use of a selective inhibitor of activated TAFI and by regression analyses we found that fibrinolysis resistance in obese samples was attributable to PAI-1 increase and to enhanced TAFI activation. The ratio between the circulating levels of D-dimer and thrombin-antithrombin complex, a marker of in vivo fibrinolysis, was significantly lower in obese children, suggesting a reduced fibrinolytic efficiency. These data indicate that paediatric obesity is associated with a hypofibrinolytic state which might contribute to the increased thrombotic risk associated with this condition.

Interactions of PEO–PPO–PEO block copolymers with lipid membranes: a computational and experimental study linking membrane lysis with polymer structure

Poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) block co-polymers (PEO–PPO–PEO, sold as Pluronics, Poloxamers, Tetronics) are a widely used class of amphiphilic materials for different biological applications. In fact for certain members of the Pluronics series, the interactions of block segments with living cells alter the lipid membrane properties and facilitate the permeation of drugs. A fuller understanding of the molecular mechanisms underpinning these interactions is essential for ensuring their safety and efficacy in biomedical applications and to inform the design of new amphiphilic copolymers for potential use in a clinical setting. In this paper, by means of atomistic molecular dynamics simulations and membrane lysis assays, we investigate the relationship between the molecular conformations of a subset of the Pluronic copolymers (L31, L61, L62 and L64) and their haemolytic activity. Our computational studies suggest that the hydrophilic blocks in these copolymers interact with the polar head groups of lipid molecules, resulting in a predicted modification of the structure of the membranes. Parallel membrane lysis assays in human erythrocytes indicate differences in the rates of haemolysis, as a result of incubation with these polymers, that correlate well with the predicted interactions from the atomistic simulations. The computational data thus provide a putative mechanism to rationalize the available experimental data on membrane lysis by these copolymers and quantitatively agree with haemoglobin release endpoints measured when copolymers with the same molecular weight and structure as of those modelled are incubated with erythrocytes. The data further suggest some new structure–function relationships at the nanoscale that are likely to be of importance in determining the biological activity of these otherwise inert copolymers.

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria.

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Rat and Mouse CD94 Associate Directly with the Activating Transmembrane Adaptor Proteins DAP12 and DAP10 and Activate NK Cell Cytotoxicity

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.