ABSTRACT
The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.
Subject(s)
Anion Exchange Protein 1, Erythrocyte , Ankyrins , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/chemistry , Anion Exchange Protein 1, Erythrocyte/metabolism , Ankyrins/metabolism , Cytoskeletal Proteins/metabolism , Erythrocyte Membrane/chemistry , Erythrocyte Membrane/metabolism , Erythrocytes/metabolism , Humans , SpectrinABSTRACT
The new coronavirus SARS-CoV-2, first identified in Wuhan, China in December, 2019, can cause Severe Acute Respiratory Syndrome (SARS) with massive alveolar damage and progressive respiratory failure. We present the relevant autopsy findings of the first patient known to have died from COVID19 pneumonia in Spain, carried out on the 14th of February, 2020, in our hospital (Hospital Arnau de Vilanova-Lliria, Valencia). Histological examination revealed typical changes of diffuse alveolar damage (DAD) in both the exudative and proliferative phase of acute lung injury. Intra-alveolar multinucleated giant cells, smudge cells and vascular thrombosis were present. The diagnosis was confirmed by reverse real-time PCR assay on a throat swab sample taken during the patient's admission. The positive result was reported fifteen days subsequent to autopsy.
Subject(s)
Autopsy , Betacoronavirus , Coronavirus Infections/pathology , Lung/pathology , Pandemics , Pneumonia, Viral/pathology , Respiratory Distress Syndrome/etiology , Aged , Alveolar Epithelial Cells/ultrastructure , Anion Exchange Protein 1, Erythrocyte/analysis , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Carcinoma, Transitional Cell/complications , China , Clinical Laboratory Techniques , Community-Acquired Infections/diagnosis , Coronavirus Infections/complications , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , DNA-Binding Proteins/analysis , Humans , Lung/virology , Macrophages/chemistry , Macrophages/ultrastructure , Male , Pneumonia/diagnosis , Pneumonia, Viral/complications , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Respiratory Distress Syndrome/pathology , SARS-CoV-2 , Spain/epidemiology , Transcription Factors/analysis , Travel , Urinary Bladder Neoplasms/complicationsABSTRACT
OBJECTIVES: Vulvar squamous cell carcinoma (vSCC) is a gynecologic malignancy diagnosed in nearly 4500 women in the United States each year. Current criteria for treatment planning provide inadequate assessment of aggressive vSCC cases, resulting in insufficient use of adjuvant treatments and high rates of vSCC recurrence. Perineural invasion (PNI) is a pathologic feature inconsistently included in the assessment of vSCC, because its relevance to clinical outcomes in these women is not well defined. The purpose of this study was to determine the association between PNI and relevant clinical parameters such as recurrence. METHODS: A total of 103 cases of vSCC were evaluated for PNI using pathology report review and immunohistochemistry dual-chromogen staining for S100 and AE1/3. Medical records were reviewed for clinical and follow-up data. Data were analyzed using univariate and multivariate logistic regression statistical methods. RESULTS: Patients with vSCC containing PNI had a greater risk for cancer recurrence than those whose tumors did not contain PNI (odds ratio=2.8, P=0.0290). There was no significant correlation between the presence of PNI and nodal involvement, stage, or lymphovascular invasion. Tumors with PNI had greater depth of invasion (DOI) (P=0.0047); however, DOI was not associated with recurrence (P=0.2220). When analyzed using a multivariable logistic regression model, PNI was an independent predictor of recurrence in vSCC (adjusted odds ratio=2.613, P=0.045). CONCLUSIONS: PNI is an independent indicator of risk for recurrence in vSCC. The association of PNI with increased risk for recurrence, independent of DOI, nodal involvement, lymphovascular invasion, or stage, should encourage practicing pathologists to thoroughly search for and report the presence of PNI in vSCC.
Subject(s)
Carcinoma, Squamous Cell/pathology , Neoplasm Recurrence, Local , Peripheral Nerves/pathology , Vulvar Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Anion Exchange Protein 1, Erythrocyte/analysis , Antiporters/analysis , Biomarkers, Tumor/analysis , Biopsy , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/therapy , Female , Humans , Immunohistochemistry , Logistic Models , Middle Aged , Multivariate Analysis , Neoplasm Invasiveness , Neoplasm Staging , Odds Ratio , Retrospective Studies , Risk Factors , S100 Proteins/analysis , Treatment Outcome , Vulvar Neoplasms/chemistry , Vulvar Neoplasms/therapy , Young AdultABSTRACT
The renal ammonium transporter RhBG and anion exchanger 1 kAE1 colocalize in the basolateral domain of α-intercalated cells in the distal nephron. Although we have previously shown that RhBG is linked to the spectrin-based skeleton through ankyrin-G and that its NH3 transport activity is dependent on this association, there is no evidence for an interaction of kAE1 with this adaptor protein. We report here that the kAE1 cytoplasmic N terminus actually binds to ankyrin-G, both in yeast two-hybrid analysis and by coimmunoprecipitation in situ in HEK293 cells expressing recombinant kAE1. A site-directed mutagenesis study allowed the identification of three dispersed regions on kAE1 molecule linking the third and fourth repeat domains of ankyrin-G. One secondary docking site corresponds to a major interacting loop of the erythroid anion exchanger 1 (eAE1) with ankyrin-R, whereas the main binding region of kAE1 does not encompass any eAE1 determinant. Stopped flow spectrofluorometry analysis of recombinant HEK293 cells revealed that the Cl(-)/HCO3 (-) exchange activity of a kAE1 protein mutated on the ankyrin-G binding site was abolished. This disruption impaired plasma membrane expression of kAE1 leading to total retention on cytoplasmic structures in polarized epithelial Madin-Darby canine kidney cell transfectants. kAE1 also directly interacts with RhBG without affecting its surface expression and NH3 transport function. This is the first description of a structural and functional RhBG·kAE1·ankyrin-G complex at the plasma membrane of kidney epithelial cells, comparable with the well known Rh·eAE1·ankyrin-R complex in the red blood cell membrane. This renal complex could participate in the regulation of acid-base homeostasis.
Subject(s)
Ammonium Compounds/metabolism , Anion Exchange Protein 1, Erythrocyte/metabolism , Ankyrins/metabolism , Epithelial Cells/metabolism , Glycoproteins/metabolism , Kidney/cytology , Membrane Transport Proteins/metabolism , Animals , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/genetics , Ankyrins/analysis , Binding Sites , Cell Line , Dogs , Glycoproteins/analysis , HEK293 Cells , Humans , Membrane Transport Proteins/analysis , Mutagenesis, Site-Directed , Protein Interaction Mapping , Protein Interaction MapsABSTRACT
A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.
Subject(s)
Actin Cytoskeleton/chemistry , Erythrocyte Membrane/chemistry , Securin/deficiency , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Animals , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/metabolism , Blotting, Western , Cytoskeletal Proteins/analysis , Cytoskeletal Proteins/metabolism , Erythrocyte Membrane/metabolism , Erythrocyte Membrane/ultrastructure , Fatty Acids, Unsaturated/analysis , Fatty Acids, Unsaturated/metabolism , Membrane Lipids/analysis , Membrane Lipids/metabolism , Membrane Proteins/analysis , Membrane Proteins/metabolism , Mice , Mice, Knockout , Microscopy , Monosaccharide Transport Proteins/analysis , Monosaccharide Transport Proteins/metabolism , Palmitic Acid/analysis , Palmitic Acid/metabolism , Plant Lectins/chemistry , Securin/genetics , Spectrin/analysis , Spectrin/metabolismABSTRACT
Resveratrol (Res) is a naturally occurring phytoalexin with apoptotic and inducing-glob effects in leukemic cells, but the potential induction of erythroid differentiation in cells is not fully understood. Here, we investigated the effects of Res on human erythro-megakaryoblastic leukemia cell line K562. Among the treated cells, proliferation was inhibited and the occurrence of cell apoptosis and cell death were detected. Erythroid differentiation assay was explored, and we found that Res could increase the expression of glycophorin A (GPA), HBA1, HBB, and γ-globin genes and enforced the expression of GPA, CD71, and Band3 proteins. Res also induced K562 cell autophagy when the concentration of Res was increased up to 50 or 100 µM. Our findings suggested that Res possesses the potency not only inducing apoptosis but also inducing erythroid differentiation and autophagy in K562 cells. These results provide that Res may be a therapeutic candidate for chronic myelogenous leukemia treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Erythroid Cells/drug effects , Stilbenes/pharmacology , Anion Exchange Protein 1, Erythrocyte/analysis , Antigens, CD/analysis , Cell Differentiation/drug effects , Cell Proliferation , Erythroid Cells/cytology , Glycophorins/analysis , Humans , K562 Cells , Receptors, Transferrin/analysis , ResveratrolABSTRACT
CONTEXT: Aldosterone (Aldo) effects include NADPH oxidase activation involved in Aldo-related oxidative stress. Red blood cells (RBCs) are particularly sensitive to oxidative assault, and both the formation of high molecular weight aggregates (HMWAs) and the diamide-induced Tyr phosphorylation (Tyr-P) level of membrane band 3 can be used to monitor their redox status. OBJECTIVE: The Aldo-related alterations in erythrocytes were evaluated by comparing in vitro evidence. DESIGN: This was a multicenter comparative study. STUDY PARTICIPANTS: The study included 12 patients affected by primary aldosteronism (PA) and 6 healthy control subjects (HCs), whose RBCs were compared with those of patients with PA. For in vitro experiments, RBCs from HCs were incubated with increasing Aldo concentrations. MAIN OUTCOME MEASURES: The Tyr-P level, band 3 HMWA formation, and autologous IgG binding were evaluated. RESULTS: In patients with PA, both Tyr-P levels and band 3 HMWAs were higher than those in HCs. RBCs from HCs were treated with increasing Aldo concentrations in both platelet-poor plasma (PPP) and charcoal-stripped (CS)-PPP. Results showed that Aldo had dose- and time-dependent effects on band 3 Tyr-P and HMWA formation in CS-PPP more than in PPP. These effects were almost completely prevented by canrenone or cortisol. Aldo-related membrane alterations led to increased autologous IgG binding. CONCLUSIONS: Erythrocytes from patients with PA show oxidative-like stress evidenced by increased HMWA content and diamide-induced band 3 Tyr-P level. Aldo effects are mediated by the mineralocorticoid receptor, as suggested by the inhibitory effects of canrenone, an antagonist of Aldo. In CS-PPP, in which Aldo induces remarkable membrane alterations leading to IgG binding, Aldo may be responsible for premature RBC removal from circulation.
Subject(s)
Erythrocytes/metabolism , Hyperaldosteronism/blood , Adult , Aged , Anion Exchange Protein 1, Erythrocyte/analysis , Diamide/pharmacology , Erythrocyte Aggregation , Female , Humans , Immunoglobulin G/metabolism , Male , Middle Aged , Oxidative StressABSTRACT
Terminal erythroid differentiation starts from morphologically recognizable proerythroblasts that proliferate and differentiate to generate red cells. Although this process has been extensively studied in mice, its characterization in humans is limited. By examining the dynamic changes of expression of membrane proteins during in vitro human terminal erythroid differentiation, we identified band 3 and α4 integrin as optimal surface markers for isolating 5 morphologically distinct populations at successive developmental stages. Functional analysis revealed that these purified cell populations have distinct mitotic capacity. Use of band 3 and α4 integrin enabled us to isolate erythroblasts at specific developmental stages from primary human bone marrow. The ratio of erythroblasts at successive stages followed the predicted 1:2:4:8:16 pattern. In contrast, bone marrows from myelodysplastic syndrome patients exhibited altered terminal erythroid differentiation profiles. Thus, our findings not only provide new insights into the genesis of the red cell membrane during human terminal erythroid differentiation but also offer a means of isolating and quantifying each developmental stage during terminal erythropoiesis in vivo. Our findings should facilitate a comprehensive cellular and molecular characterization of each specific developmental stage of human erythroblasts and should provide a powerful means of identifying stage-specific defects in diseases associated with pathological erythropoiesis.
Subject(s)
Erythroblasts/cytology , Erythropoiesis , Anion Exchange Protein 1, Erythrocyte/analysis , Antigens, CD34/analysis , Bone Marrow Cells/cytology , Bone Marrow Cells/pathology , Cell Separation/methods , Cells, Cultured , Cytoskeletal Proteins/analysis , Erythroblasts/pathology , Flow Cytometry/methods , Humans , Immunoblotting , Integrin alpha4/analysis , Membrane Proteins/analysis , Mitosis , Myelodysplastic Syndromes/pathologyABSTRACT
There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388-911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3 mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment with the endoglycosidase, PNGase F, showed that AE1MD-Rho is not N-glycosylated. AE1MD-Rho solubilized from yeast membranes, with Fos-choline detergent, was purified to 93% homogeneity in a single-step, using a 1D4 antibody affinity resin, in amounts up to 2.5 mg from 18 l of culture. The ability of purified AE1MD-Rho to transport sulfate was examined in reconstituted vesicles. The rate of sulfate efflux mediated by vesicles reconstituted with AE1MD-Rho was indistinguishable from vesicles with purified erythrocyte-source AE1. Using this purification strategy, sufficient amounts of functional, homogeneous AE1MD-Rho can be purified to enable crystallization trials.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/genetics , Anion Exchange Protein 1, Erythrocyte/isolation & purification , Saccharomyces cerevisiae/genetics , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/metabolism , Anions/metabolism , Base Sequence , Cloning, Molecular , Detergents , Genetic Vectors , Glycosylation , Humans , Molecular Sequence Data , Rhodopsin/analysis , Rhodopsin/genetics , Rhodopsin/isolation & purification , Rhodopsin/metabolism , Saccharomyces cerevisiae/ultrastructure , Up-RegulationABSTRACT
The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.
Subject(s)
Anion Exchange Protein 1, Erythrocyte/physiology , Membrane Proteins/physiology , Podocytes/metabolism , Adult , Albuminuria/metabolism , Amino Acid Sequence , Animals , Anion Exchange Protein 1, Erythrocyte/analysis , Cells, Cultured , Female , Fluorescent Antibody Technique , Humans , Kidney Glomerulus/pathology , Membrane Proteins/genetics , Mice , Molecular Sequence Data , Protein Serine-Threonine Kinases/physiology , Two-Hybrid System Techniques , XenopusABSTRACT
BACKGROUND/AIM: Identification and characterization of membrane proteins is a crucial challenge in proteomics research. Thus, we designed a novel method to prepare proteins possessing extensive hydrophobic stretches for mass spectrometry studies, without sacrificing other classes of proteins. MATERIALS AND METHODS: This method uses sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and relies solely on a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) instrument, the most common and easiest to use mass spectrometer. RESULTS: Using this analytical procedure, a significant number of hydrophobic peptides were recovered, with no reduction in overall sequence coverage and with a good identification of transmembrane proteins sequence. Applying this method to the systematic identification of proteins located in lipid rafts, up to 47% of identified proteins were obtained with an improvement of sequence coverage. CONCLUSION: The procedure presented here is suitable for both identifying purified hydrophobic proteins and systematically investigating hydrophobic protein mixtures. It can be easily applied even in non-dedicated laboratories, such as those mostly devoted to clinical chemistry.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Membrane Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Actins/analysis , Animals , Anion Exchange Protein 1, Erythrocyte/analysis , Hydrophobic and Hydrophilic Interactions , Mice , Myosin Light Chains/analysis , Proteomics/methods , RatsABSTRACT
Pleomorphic carcinoma of the breast is considered a rare variant of high-grade ductal NOS carcinoma (NOS-IDC), and the prognosis is poor. However, its clinicopathologic features are not well-characterized. Using the criteria delineated in the World Health Organization breast tumor classification of 2003, ten cases of pleomorphic carcinoma were identified from 9794 NOS-IDC in our archived materials that were originally diagnosed as high-grade infiltrating ductal carcinoma of breast. To investigate the clinicopathologic characteristics and to elucidate the histologic diagnosis and differential diagnosis of this entity, we reviewed the pathology manifestations and clinical features of these cases and examined the tumor expression of ER, PR, PCNA, AE(1)/AE(3), p53, S-100, C-erbB-2, EMA, p63, and Bcl-2 by immunohistochemistry.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Adult , Aged , Anion Exchange Protein 1, Erythrocyte/analysis , Antiporters/analysis , Female , Humans , Immunohistochemistry , Membrane Proteins/analysis , Middle Aged , Mucin-1/analysis , Proliferating Cell Nuclear Antigen/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , Receptor, ErbB-2/analysis , Receptors, Estrogen/analysis , Retrospective Studies , S100 Proteins/analysis , Tumor Suppressor Protein p53/analysisABSTRACT
Quantification of immunostaining is a widely used technique in pathology. Nonetheless, techniques that rely on human vision are prone to inter- and intraobserver variability, and they are tedious and time consuming. Digital image analysis (DIA), now available in a variety of platforms, improves quantification performance: however, the stability of these different DIA systems is largely unknown. Here, we describe a method to measure the reproducibility of DIA systems. In addition, we describe a new image-processing strategy for quantitative evaluation of immunostained tissue sections using DAB/hematoxylin-stained slides. This approach is based on image subtraction, using a blue low pass filter in the optical train, followed by digital contrast and brightness enhancement. Results showed that our DIA system yields stable counts, and that this method can be used to evaluate the performance of DIA systems. The new image-processing approach creates an image that aids both human visual observation and DIA systems in assessing immunostained slides, delivers a quantitative performance similar to that of bright field imaging, gives thresholds with smaller ranges, and allows the segmentation of strongly immunostained areas, all resulting in a higher probability of representing specific staining. We believe that our approach offers important advantages to immunostaining quantification in pathology.
Subject(s)
Image Processing, Computer-Assisted/methods , Immunohistochemistry/methods , Pathology/methods , Anion Exchange Protein 1, Erythrocyte/analysis , Antiporters/analysis , Carcinoma, Squamous Cell/pathology , Humans , Ki-67 Antigen/analysis , Reproducibility of ResultsABSTRACT
A 57-year-old woman with splenomegaly, moderate anemia and a paraspinal mass was found to have strikingly increased erythropoiesis, which was left shifted and dysplastic with many multinucleated cells. An increased percentage of CD34-positive and CD117-positive cells suggested a diagnosis of pure erythroleukemia. Further investigations were performed.
Subject(s)
Antigens, CD34/analysis , Leukemia, Erythroblastic, Acute/diagnosis , Proto-Oncogene Proteins c-kit/analysis , Anion Exchange Protein 1, Erythrocyte/analysis , Blood Protein Electrophoresis , Bone Marrow Examination , Diagnosis, Differential , Diagnostic Errors/prevention & control , Electrophoresis, Polyacrylamide Gel , Female , Flow Cytometry , Giant Cells/pathology , Humans , Immunohistochemistry , Immunophenotyping , Leukemia, Erythroblastic, Acute/metabolism , Middle Aged , Splenomegaly/pathologyABSTRACT
Se estandarizó un método inmunoenzimático (ELISA) para la determinación de anticuerpos naturales anti banda 3 acoplando a placas Chemobond la proteína banda 3 obtenida a partir de la membrana de eritrocitos humanos mediante una combinación de cromatografía de intercambio aniónico y afinidad. Se emplearon 3 mg de la proteína banda 3 por pozo, lográndose un nivel de detección de 1 mg/mL de anticuerpo anti banda 3. Los resultados indican la utilidad del método para su empleo en la medición de los niveles de anticuerpos naturales anti banda 3 en pacientes con drepanocitosis, tanto en estado basal como en las crisis vasooclusivas dolorosas(AU)
An immunoenzimatic assay (ELISA) was standardized to determine the natural anti-band 3 antibodies by coupling with Chemobond plates the band 3 protein obtained from the membrane of human erythrocytes by a combination of affinity and anion exchange chromatography. 3 g of band 3 protein per well were used. A level of detection of 1 g/mL of anti-band 3 antibody was achieved. The results showed the usefulness of the method to measure the levels of natural anti-band 3 antibodies in patients with drepanocytes, both in basal state and in the painful vasocclusive crises(AU)
Subject(s)
Humans , Male , Female , Adult , Enzyme-Linked Immunosorbent Assay/methods , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/chemistryABSTRACT
Se estandarizó un método inmunoenzimático (ELISA) para la determinación de anticuerpos naturales anti banda 3 acoplando a placas Chemobond la proteína banda 3 obtenida a partir de la membrana de eritrocitos humanos mediante una combinación de cromatografía de intercambio aniónico y afinidad. Se emplearon 3 mg de la proteína banda 3 por pozo, lográndose un nivel de detección de 1 mg/mL de anticuerpo anti banda 3. Los resultados indican la utilidad del método para su empleo en la medición de los niveles de anticuerpos naturales anti banda 3 en pacientes con drepanocitosis, tanto en estado basal como en las crisis vasooclusivas dolorosas.
An immunoenzimatic assay (ELISA) was standardized to determine the natural anti-band 3 antibodies by coupling with Chemobond plates the band 3 protein obtained from the membrane of human erythrocytes by a combination of affinity and anion exchange chromatography. 3 g of band 3 protein per well were used. A level of detection of 1 g/mL of anti-band 3 antibody was achieved. The results showed the usefulness of the method to measure the levels of natural anti-band 3 antibodies in patients with drepanocytes, both in basal state and in the painful vasocclusive crises.
Subject(s)
Humans , Male , Adult , Female , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/chemistry , Enzyme-Linked Immunosorbent Assay/methodsABSTRACT
Our aim was to evaluate red blood cell (RBC) membrane protein composition in chronic kidney disease (CKD) stage 5 patients under haemodialysis (HD) and recombinant human erythropoietin (rhEPO) therapy, and its linkage to rhEPO hyporesponsiveness. We evaluated in 63 CKD stage 5 patients (32 responders and 31 non-responders to rhEPO therapy) and in 26 healthy controls RBC count, haematocrit, haemoglobin concentration, haematimetric indices, reticulocyte count, reticulocyte production index, RBC osmotic fragility test and membrane protein analyses. CKD stage 5 patients presented significant changes in membrane protein composition, namely a reduction in spectrin, associated to altered protein 4.1/spectrin and spectrin/band 3 ratios. Non-responder CKD stage 5 patients were more anaemic, with more microcytic and anisocytic RBCs, than responders; significantly altered ankyrin/band 3 and spectrin/ankyrin ratios were also observed. CKD stage 5 patients under HD are associated with an altered protein membrane structure, which seems to the disease itself and/or to the interaction with HD membranes.
Subject(s)
Anemia/blood , Blood Proteins/analysis , Erythrocyte Membrane/chemistry , Erythropoietin/analogs & derivatives , Erythropoietin/therapeutic use , Kidney Failure, Chronic/blood , Membrane Proteins/blood , Renal Dialysis , Aged , Anemia/drug therapy , Anemia/etiology , Anion Exchange Protein 1, Erythrocyte/analysis , Ankyrins/analysis , Darbepoetin alfa , Diabetic Nephropathies/blood , Diabetic Nephropathies/therapy , Drug Resistance , Epoetin Alfa , Female , Folic Acid/therapeutic use , Humans , Iron/therapeutic use , Kidney Failure, Chronic/complications , Kidney Failure, Chronic/therapy , Male , Membranes, Artificial , Middle Aged , Oxidation-Reduction , Recombinant Proteins , Renal Dialysis/adverse effects , Renal Dialysis/instrumentation , Spectrin/analysisABSTRACT
Malignant rhabdoid tumor, first described in the kidney of young infants, is a rare and highly aggressive neoplasm of controversial histogenesis that has been reported at many other sites, including the gastrointestinal tract. However, malignant rhabdoid tumor of the small intestine is very rare, with only seven cases published to date. We report a 70-year-old man who presented with abdominal pain and weight loss, and showed a perforated jejunal mass with disseminated metastases by imaging. The patient underwent partial jejunectomy and biopsy of a liver metastasis. Microscopically, the tumor was characterized by neoplastic cells with vesicular nuclei, large nucleoli and abundant eccentric cytoplasm with hyaline globular intracytoplasmic inclusions. Immunohistochemically, the neoplasm coexpressed vimentin and epithelial antigens (AE1/AE3, Cam 5.2, CK34betaE12, CK19 and EMA), most of them showing a peculiar immunostaining pattern in relation to the globular inclusions. Ultrastructurally, the inclusions corresponded to paranuclear whorls of intermediate filaments. The patient received postoperative chemotherapy but died 9 months after surgery. In summary, we report the exceptional case of an undifferentiated carcinoma of the jejunum with rhabdoid phenotype. As with tumors at other sites, recognition of rhabdoid morphology in small intestine neoplasms is of significance because the prognosis is extremely poor.
Subject(s)
Carcinoma/pathology , Jejunal Neoplasms/pathology , Liver Neoplasms/secondary , Rhabdoid Tumor/pathology , Aged , Anion Exchange Protein 1, Erythrocyte/analysis , Anion Exchange Protein 1, Erythrocyte/metabolism , Biomarkers/analysis , Biomarkers/metabolism , Biopsy , Carcinoma/metabolism , Carcinoma/therapy , Cell Nucleolus/pathology , Fatal Outcome , Humans , Hyalin/metabolism , Immunohistochemistry , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Jejunal Neoplasms/metabolism , Jejunal Neoplasms/therapy , Jejunum/metabolism , Jejunum/pathology , Keratins/analysis , Keratins/metabolism , Liver/pathology , Liver Neoplasms/pathology , Male , Neoplasm Proteins/analysis , Neoplasm Proteins/metabolism , Rhabdoid Tumor/metabolism , Rhabdoid Tumor/therapy , Vimentin/analysis , Vimentin/metabolismABSTRACT
Primary cutaneous carcinosarcomas (PCCs) are rare malignant neoplasms that are characterized by biphasic epithelial and mesenchymal differentiation. When the biphasic nature is not evident, immunohistochemical studies may be important in the diagnosis of PCCs. Although AE1/AE3 is frequently used to demonstrate the epithelial component, it may not be strongly expressed in epithelial cells that are not well-differentiated. p63 is a protein homologue of p53 that is expressed in poorly differentiated epithelial cells. We report 3 cases of PCC. The clearly epithelial areas of each tumor were frequently positive for both markers, whereas the sarcomatous areas were negative for both markers. Epithelial cells that were poorly differentiated and not easily identifiable were positive for p63 but negative for AE1/AE3. Of interest, transitional areas showed positivity for p63 alone. These 3 cases suggest that the use of both p63 and routine cytokeratin markers such as AE1/AE3 can increase the sensitivity for distinguishing epithelial cells over a range of differentiation states, which we propose will aid in the diagnosis of PCCs. In addition, the staining pattern of AE1/AE3 and p63 in these cases further supports the conversion theory of PCC.
Subject(s)
Carcinosarcoma/pathology , DNA-Binding Proteins/analysis , Skin Neoplasms/pathology , Trans-Activators/analysis , Tumor Suppressor Proteins/analysis , Aged , Aged, 80 and over , Anion Exchange Protein 1, Erythrocyte/analysis , Antiporters/analysis , Biomarkers, Tumor/analysis , Cell Differentiation , Epithelial Cells/pathology , Humans , Keratins/analysis , Male , Middle Aged , Transcription Factors , Vimentin/analysisABSTRACT
We analyzed erythrocyte glycoconjugates in two families with congenital dyserythropoietic anemia type II (CDA-II): family 2 with the typical localization of the disease gene to chromosome 20q11.2 and family 1 in which this localization was excluded. Despite the different genetics, the erythrocyte glycoconjugate abnormalities in the two families were identical suggesting a complex inheritance of CDA-II. We also found that erythrocyte anion exchanger 1 protein is decreased in CDA-II homozygotes and obligate carriers alike.