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Focuses on the reaction of murine monoclonal antibody against Kx protein and also with beta-spectrin. Capability of murine monoclonal antibody in red cell deficiency; Characteristics of murine monoclonal antibody; Examination on the read cells of the patients.

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca 2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2 V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2 V617F , with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2 V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2 V617F , calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

Desmoid-type fibromatosis (DTF) is a very rare variant of papillary thyroid carcinoma (PTC). It is essentially a dual tumor with a component of classical PTC with malignant epithelial proliferation ( BRAF -mutated) and another component of mesenchymal proliferation ( CTNNB1 -mutated). We conducted a literature review on PTC-DTF. In total, 31 articles were identified, that together reported on 54 patients. The mean age was 47 years, with a 2.2:1 female predominance. No ultrasound features were found to be helpful in differentiating PTC-DTF from other PTC variants. Of the 43 cases that reported histological details, 60% had locally infiltrative disease (T3b or T4). Around 48% had cervical lymph node metastases, but none had distant metastases. While PTC-DTF may be locally more aggressive than classic PTC, its overall behavior is similar and can include extrathyroidal extension and lymph node metastases, which may contain a stromal component and show extranodal invasion. The mainstay of treatment for PTC-DTF is surgery, and the DTF component is not expected to be sensitive to radioactive iodine. External radiotherapy, non-steroidal anti-inflammatory drugs, tyrosine kinase inhibitors and chemotherapy have also been used in selected cases. Due to the rarity of these tumors and the lack of specific treatment guidelines, management should be discussed in a multidisciplinary team.

Vaso-occlusive crises are the hallmark of sickle cell disease (SCD). They are believed to occur in two steps, starting with adhesion of deformable low-dense red blood cells (RBCs), or other blood cells such as neutrophils, to the wall of post-capillary venules, followed by trapping of the denser RBCs or leukocytes in the areas of adhesion because of reduced effective lumen-diameter. In SCD, RBCs are heterogeneous in terms of density, shape, deformability and surface proteins, which accounts for the differences observed in their adhesion and resistance to shear stress. Sickle RBCs exhibit abnormal adhesion to laminin mediated by Lu/BCAM protein at their surface. This adhesion is triggered by Lu/BCAM phosphorylation in reticulocytes but such phosphorylation does not occur in mature dense RBCs despite firm adhesion to laminin. In this study, we investigated the adhesive properties of sickle RBC subpopulations and addressed the molecular mechanism responsible for the increased adhesion of dense RBCs to laminin in the absence of Lu/BCAM phosphorylation. We provide evidence for the implication of oxidative stress in post-translational modifications of Lu/BCAM that impact its distribution and cis-interaction with glycophorin C at the cell surface activating its adhesive function in sickle dense RBCs.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

Tumor cell migration depends on the interactions of adhesion proteins with the extracellular matrix. Lutheran/basal cell adhesion molecule (Lu/BCAM) promotes tumor cell migration by binding to laminin α5 chain, a subunit of laminins 511 and 521. Lu/BCAM is a type I transmembrane protein with a cytoplasmic domain of 59 (Lu) or 19 (Lu(v13)) amino acids. Here, using an array of techniques, including site-directed mutagenesis, immunoblotting, FRET, and proximity-ligation assays, we show that both Lu and Lu(v13) form homodimers at the cell surface of epithelial cancer cells. We mapped two small- XXX -small motifs in the transmembrane domain as potential sites for monomers docking and identified three cysteines in the cytoplasmic domain as being critical for covalently stabilizing dimers. We further found that Lu dimerization and phosphorylation of its cytoplasmic domain were concomitantly needed to promote cell migration. We conclude that Lu is the critical isoform supporting tumor cell migration on laminin 521 and that the Lu:Lu(v13) ratio at the cell surface may control the balance between cellular firm adhesion and migration., (© 2019 Guadall et al.)

Spleen dysfunction is central to morbidity and mortality in children with sickle cell anemia (SCA). The initiation and determinants of spleen injury, including acute splenic sequestration (ASS) have not been established. We investigated splenic function longitudinally in a cohort of 57 infants with SCA enrolled at 3 to 6 months of age and followed up to 24 months of age and explored the respective contribution of decreased red blood cell (RBC) deformability and increased RBC adhesion on splenic injury, including ASS. Spleen function was evaluated by sequential 99m Tc heated RBC spleen scintigraphy and high-throughput quantification of RBCs with Howell-Jolly bodies (HJBs). At 6 and 18 months of age, spleen filtration function was decreased in 32% and 50% of infants, respectively, whereas the median %HJB-RBCs rose significantly (from 0.3% to 0.74%). An excellent correlation was established between %HJB-RBCs and spleen scintigraphy results. RBC adhesion to laminin and endothelial cells increased with time. Adhesion to endothelial cells negatively correlated with splenic function. Irreversibly sickled cells (ISCs), used as a surrogate marker of impaired deformability, were detected at enrollment and increased significantly at 18 months. %ISCs correlated positively with %HJB-RBCs and negatively with splenic uptake, indicating a relationship between their presence in the circulation and spleen dysfunction. In the subgroup of 8 infants who subsequently experienced ASS, %ISCs at enrollment were significantly higher compared with the asymptomatic group, suggesting a major role of impaired deformability in ASS. Higher levels of %HJB-RBCs were observed after the occurrence of ASS, demonstrating its negative impact on splenic function., (© 2019 by The American Society of Hematology.)

Reticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes., (© 2018 by The American Society of Hematology.)

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms., (Copyright © 2018 Ferrata Storti Foundation.)

The protein toxin Cytotoxic Necrotizing Factor 1 (CNF1) is a major virulence factor of pathogenic Escherichia coli strains. It belongs to a family of single chain AB-toxins, which enter mammalian cells by receptor-mediated endocytosis. Recently, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as a cellular receptor for CNF1. Here, we identified the Ig-like domain 2 of Lu/BCAM as main interaction site of the toxin by direct protein-protein interaction and competition studies. Using surface plasmon resonance, we showed a high affinity CNF-Lu/BCAM interaction with a KD of 2.8 nM. Furthermore, we performed small-angle X-ray scattering to define the molecular envelope of the Lu/BCAM-CNF1 complex, suggesting a 6:1 ratio of Lu/BCAM to CNF1 in the receptor-toxin complex. This study leads to a deeper understanding of the interaction between CNF1 and Lu/BCAM, and presents novel opportunities for the development of future anti-toxin strategies., Competing Interests: The authors declare no conflict of interest.

In order for Staphylococcus aureus to thrive inside the mammalian host, the bacterium has to overcome iron scarcity. S. aureus is thought to produce toxins that lyse erythrocytes, releasing hemoglobin, the most abundant iron source in mammals. Here we identify the Duffy antigen receptor for chemokines (DARC) as the receptor for the S. aureus hemolytic leukocidins LukED and HlgAB. By assessing human erythrocytes with DARC polymorphisms, we determined that HlgAB- and LukED-mediated lysis directly relates to DARC expression. DARC is required for S. aureus-mediated lysis of human erythrocytes, and DARC overexpression is sufficient to render cells susceptible to toxin-mediated lysis. HlgA and LukE bind directly to DARC through different regions, and by targeting DARC, HlgAB and LukED support S. aureus growth in a hemoglobin-acquisition-dependent manner. These findings elucidate how S. aureus targets and lyses erythrocytes to release one of the scarcest nutrients within the mammalian host., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

Background: Decisions regarding Cardio-Pulmonary Resuscitation (CPR) and Do Not Attempt Resuscitation (DNAR) orders remain demanding, as does including patients in the process., Objectives: To explore physicians' justification for CPR/DNAR orders and decisions regarding patient inclusion, as well as their reports of how they initiated discussions with patients., Methods: We administered a face-to-face survey to residents in charge of 206 patients including DNAR and CPR orders, with or without patient inclusion., Results: Justifications were provided for 59% of DNAR orders and included severe comorbidity, patients and families' resuscitation preferences, patients' age, or poor prognosis or quality of life. Reasons to include patients in CPR/DNAR decisions were provided in 96% and 84% of cases, and were based on respect for autonomy, clinical assessment of the situation as not too severe, and the view that such inclusion was required. Reasons for not including patients were offered in 84% of cases for CPR and in 70% for DNAR. They included absent decision-making capacity, a clinical situation viewed as good (CPR) or offering little hope of recovery (DNAR), barriers to communication, or concern that discussions could be emotionally difficult or superfluous. Decisions made earlier in the patient's management were infrequently viewed as requiring revision. Residents reported a variety of introductions to discussions with patients., Conclusions: These results provide better understanding of reasons for CPR/DNAR decisions, reasons for patient inclusion or lack thereof, and ways in which such inclusion is initiated. They also point to potential side-effects of implementing CPR/DNAR recommendations without in-depth and practical training. This should be part of a regular audit and follow-up process for such recommendations.

Although anal canal squamous cell carcinoma is rare, the general practitioner should consider this diagnosis in a patient with persistent lower abdominal symptoms. While classically observed in older women, an increased incidence is also seen in HIV-positive patients or patients with a history of human papillomavirus infection. Initial diagnosis and local work-up require assessment by a proctologist. Standard curative treatment combines radiotherapy with 5-FU- and MMC-based chemotherapy. Salvage surgery should be discussed in case of local relapse. The general practitioner, the proctologist and the radiation oncologist, all participate in post-treatment surveillance.

Background: Fistulas arising from the perforation of anal cancer into adjacent organs are a debilitating complication in the course of the disease., Objective: We studied intra-arterial chemotherapy as a strategy to close such fistulas before the initiation of standard chemoradiation., Design: This study was based on a retrospective chart review., Setting: The investigation was conducted at Geneva University Hospital., Patients: Eight patients with anal cancer-related fistulas were included in the study., Intervention: Patients were treated at our institution from 2002 to 2009 with upfront chemotherapy consisting of 1 to 4 cycles of intra-arterial cisplatin, 5-fluorouracil, methotrexate, and mitomycin C, and intravenous bleomycin. Intra-arterial chemotherapy was followed by standard chemoradiation., Main Outcome Measure: Fistula closure was assessed by an expert proctologist., Results: Complete closure of fistulas was documented in 7 of 8 patients. Toxicity was manageable and consisted mainly of thrombocytopenia, neutropenia, and febrile neutropenia as well as fatigue., Limitations: This is a retrospective, uncontrolled review of only 8 patients and thus a meaningful comparison with standard chemoradiation is not feasible., Conclusion: Upfront intra-arterial chemotherapy is a promising strategy to close anal cancer-related fistulas before initiating chemoradiation, potentially obviating the need for hazardous reconstructive surgery after radiotherapy.

Objective: To determine the prevalence of cardiopulmonary resuscitation (CPR) and do-not-attempt-resuscitation (DNAR) orders, to define factors associated with CPR/DNAR orders and to explore how physicians make and document these decisions., Methods: We prospectively reviewed CPR/DNAR forms of 1,446 patients admitted to the General Internal Medicine Department of the Geneva University Hospitals, a tertiary-care teaching hospital in Switzerland. We additionally administered a face-to-face survey to residents in charge of 206 patients including DNAR and CPR orders, with or without patient inclusion., Results: 21.2% of the patients had a DNAR order, 61.7% a CPR order and 17.1% had neither. The two main factors associated with DNAR orders were a worse prognosis and/or a worse quality of life. Others factors were an older age, cancer and psychiatric diagnoses, and the absence of decision-making capacity. Residents gave four major justifications for DNAR orders: important comorbid conditions (34%), the patients' or their family's resuscitation preferences (18%), the patients' age (14.2%), and the absence of decision-making capacity (8%). Residents who wrote DNAR orders were more experienced. In many of the DNAR or CPR forms (19.8 and 16%, respectively), the order was written using a variety of formulations. For 24% of the residents, the distinction between the resuscitation order and the care objective was not clear. 38% of the residents found the resuscitation form useful., Conclusion: Patients' prognosis and quality of life were the two main independent factors associated with CPR/DNAR orders. However, in the majority of cases, residents evaluated prognosis only intuitively, and quality of life without involving the patients. The distinction between CPR/DNAR orders and the care objectives was not always clear. Specific training regarding CPR/DNAR orders is necessary to improve the CPR/DNAR decision process used by physicians., (Copyright © 2010 S. Karger AG, Basel.)

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

Ovarian cancer accounts for a minority of female cancers but remains the leading cause of death from gynaecologic cancers and the fifth leading cause of all cancer-related deaths among women. More than two thirds of cases of ovarian cancer are diagnosed once the disease becomes symptomatic, i.e. at an advanced stage. Though early detection constitutes a legitimate aim, no screening approach has yet been shown to reduce ovarian cancer mortality. In particular, ovarian imagery with endovaginal ultrasonography and serum tumor markers (CA-125) dosage performed in asymptomatic individuals have not proven their efficacy. Screening of asymptomatic women is not therefore recommended because of the limited prevalence of ovarian cancer in the general population.

Background: Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties., Methodology/principal Findings: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy., Conclusions/significance: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.

Surveillance following curative therapy for localised breast cancer has two major aims: 1) diagnosis of recurrence or a second tumor: regular interviews and physical examinations with a frequency adapted to each case, and yearly mammography (+/- ultrasonography), are the only recommended procedure. Additional screening is not indicated in asymptomatic patient. 2) Screening for complications and side effects due to treatment: tamoxifen treatment should be stopped one week prior to any surgery or long trip to avoid thromboembolic events, and any metrorrhagia should be investigated due to the risk for endometrial cancer. Osteoporosis and arthralgia are frequently observed with aromatase inhibitors.

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane., (Copyright 1998 Academic Press.)

Toxicity effects related to leached ligands from affinity sorbents that can contaminate biological preparations were investigated in the particular case of immobilized Reactive Blue-2. Initially, identification of the real chemical structure of leached dye has been done by HPLC after incubation in extreme conditions. Toxicity investigations in vitro involving several well known tests showed no toxic effects within the studied range of dye concentration. Cell cultures behaved normally when the adhesion phase was successful; polyploidy induction in human cells by the native dye and its derivatives identified as possible leached material was very similar to standard cultures. Genotoxicity studies did not evidence any toxic effect in E. Coli cultures of dyes themselves or of the same dyes after metabolic activation.

On coating reversed-phase supports with polyoxyethylene-type non-ionic surfactants, proteins are no longer retained on such supports at moderate or low ionic strength, but they are retained at high ionic strength and can be desorbed by a decreasing ionic strength gradient. These reversibly modified supports were used for hydrophobic interaction chromatography (HIC). The proteins probably interact with the polyoxyethylene tail of the non-ionic surfactant while the hydrophobic part of the surfactant anchors the surfactant to the reversed-phase support by interactions with its alkane coverage. Although the interactions between non-ionic surfactant and reversed-phase support are non-covalent and the HIC mobile phases contained no surfactant, the modified columns were stable and could be used repeatedly. A surfactant-modified reversed-phase column provided a rapid, efficient, one-step purification of a fungal aspartic proteinase from a commercial crude preparation.