Doris Kraemer

Mrnp41 (Rae1p) associates with microtubules in HeLa cells and in neurons

Mrnp41 (hRae1p) is an evolutionarily highly conserved protein, which is a potential component of mRNP particles and plays a role in nuclear mRNA export. The protein is mainly localized at the nuclear pore complex, but is also associated with distinct nuclear domains and with a meshwork of numerous small particles in the cytoplasm (Kraemer and Blobel (1997): Proc. Natl. Acad. Sci. USA 91, 1519-1523). We show that the cytoplasmic pattern of mrnp41 is sensitive to treatment with the microtubule (MT)-depolymerizing drug nocodazole which causes disappearance of mrnp41 from the cell periphery and concentration around the nucleus. By immunofluorescence we demonstrate that mrnp41 colocalizes with MT in HeLa cells and displays an MT-like distribution in cultured neurons. Association of mrnp41 with MT is further demonstrated by copurification with MT from pig brain throughout several steps of polymerization and depolymerization. Separation of MT-associated proteins (MAPs) by phosphocellulose (PC) chromatography showed copurification of mrnp41 with MAPs. These data show an association of mrnp41 with MT and, moreover, demonstrate that an intact MT system is necessary for dispersion of mrnp41-containing particles to the cellular periphery. The essential role of mrnp41 in spindle pole separation and cell cycle progression may also be related to its ability to bind to MTs.

Kidney Na+,K+-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).

Evaluation of a novel case-based training program (d3web.Train) in hematology

The new media such as the internet and digital imaging offer new opportunities in medical education. In addition to conventional lectures, we developed a case-based simulation training program of 17 hematology cases using the novel training system d3web.Train. We evaluated the assessment of this internet course by medical students, as well as their results in the hematology exam. From a group of 150 students, 47 worked through at least one case and solved 435 cases in total; in average, these students solved 9.5 cases. Eighteen different students filled in a questionnaire about the training system and 68 questionnaires about individual cases. The main results were the students found the cases very helpful (1.5±0.6 on a scale from 1=very helpful to 5=not at all), the training system very good (1.4±0.5 on a scale from 1 to 6), and want to work with it further (1.2±0.4 on a scale from 1 to 5). During the final examination, those 16 students who answered that they had solved more than 5 from the 17 cases scored significantly better (two-sided t test, p<0.01) in the hematological part of the exam than those 34 students solving 0 to 5 cases. To our knowledge, this is the first student evaluation of a case-based training program in general hematology. The d3web.Train system offers a new and great tool for creating a training program in a reasonable amount of time, because it is able to process available patient records.

Aseptic necrosis of both femoral heads as first symptom of chronic myelogenous leukemia.

Abstract. Chronic myelogenous leukemia (CML) is a disease of the elderly; in rare cases it occurs in childhood or adolescence. One complication at primary diagnosis is leukostasis, which usually causes respiratory, retinal, or central nervous symptoms. In this report we describe the case of a 24-year-old woman who developed aseptic necrosis of both femoral heads, which was successfully treated by bore holes in the femoral heads. This is a very rare complication of severe leukostasis, leading to the diagnosis of CML in this case. To our knowledge, this is the first case of an adult patient showing aseptic necrosis of femoral heads caused by leukostasis.

Cytoskeleton and Epithelial Polarity

The membrane surface of polarized epithelial cells can be divided in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton are involved in critical steps of both generation and maintenance of cell polarity. Generation of polarity is controlled by microtubules that serve as uniformly aligned and polarized cytoplasmic guiding structures for the vectorial and selective transport of Golgi-derived carrier vesicles to the apical cell surface. Targeting of membrane proteins to the basolateral cell surface does not depend on microtubules but follows the constitutive bulk flow of membranes. Once inserted into the lipid bilayer several membrane proteins such as the kidney anion exchanger 1 (AE1) and the sodium pump become immobilized at specialized microdomains of the lateral cell surface. Evidence is provided that both membrane proteins are linked via ankyrin to the spectrin-based membrane cytoskeleton that underlies the basolateral membrane domain. Linkage of these and other integral membrane proteins to the cytoskeleton may not only place them to specialized sites of the plasma membrane but may also prevent these transporters from clustering and endocytosis, thus helping them to stay at the cell surface. In search of sequence motifs involved in binding of integral membrane proteins to components of the cytoskeleton we found that the binding interface of AE1 to protein 4.1 (an actin and spectrin cross-linking protein) consists of a cluster of five amino acid residues, namely IRRRY in AE1 and LEEDY on protein 4.1. This motif may play a more general role in cytoskeleton membrane linkages.

Nucleoporin p62 Antibodies in a Case of Mixed Connective Tissue Disease

ABSTRACT Mixed connective tissue disease is an overlap syndrome characterized by features of different systemic autoimmune diseases and a high titer of U1-snRNP antibodies. We examine here the autoantibodies to nucleoporin p62 in a severe case of mixed connective tissue disease in a young male patient. Thus far, p62 antibodies have mainly been described in cases of primary biliary cirrhosis. We speculate that the presence of p62 antibodies is an indication of a poor prognosis in connective tissue disorders.

Platelet recovery and survival measured in patients by quantitative polymerase chain reaction of mitochondrial DNA.

Mitochondrial (mt) DNA markers have been identified as potential targets for the quantification of endogenous and allogeneic platelets (PLTs) in the blood of individuals who received transfusions. Our goal was to develop a routine polymerase chain reaction (PCR) assay for ex vivo monitoring of PLT survival in patients after transfusion.

Digital PCR to assess platelet recovery after stem cell transplantation

Engraftment of platelets in patients after human hematopoietic stem cell (HSC) transplantation serves as an early and relevant sign of the success of the transplantation. The time to platelet engraftment appears to be a valid predictor of the probability of complications in transplanted patients and especially for allogeneic HSC transplantation [1]. At least two different criteria of platelet engraftment, i.e. definitions according to the CIBMTR and EBMT, have been widely applied. Both criteria utilize daily platelet counts to assess the time to a threshold count of more than 20,000/μL platelets in peripheral blood, provided that platelet transfusions have been terminated for either at least three [2] (EBMT) or at least seven [3] (CIBMTR) consecutive days. Here, we describe the development of a digital PCR assay to specifically quantify graft-derived platelets versus transfused single-donor platelets. The performance of this assay was then tested with samples from allogeneic HSC transplant patients. Samples of 2 mL EDTA anticoagulated blood from patients were mixed with an equal volume of 0.9% NaCl and centrifuged at 100×g for 10 min without brake (Hettich Rotina 46S, Hettich, Tuttlingen, Germany). A final centrifugation step at 1569×g for 5 min followed. The pellet was resuspended in 500 μL phosphate buffered saline (PBS, Invitrogen Karlsruhe, Germany). The number of platelets in the PRP was adjusted with PBS to 5 × 10–2.5 × 10 to extract the mt DNA from these platelet suspensions (500 μL). The MagnaPure Large Volume protocol (MagnaPure compact, Roche Diagnostics GmbH, Mannheim, Germany) was used to extract the mt DNA. Elution volume was 50 μL. The PCR strategy was based on generic amplification primers and allele-specific probes. The probes were designed to specifically detect alleles carrying SNPs at positions 73, 195, 295, 310, 16069, 16399 and 16,516 of the non-coding regions HVR 1 and HVR 2 of the mt genome. Droplet generation, amplification and detection of PCR products were performed according to the manufacturer’s instructions (QX200 ddPCR System, Bio-Rad Laboratories GmbH, München, Germany) using 5 μL extracted DNA. Specificity of the assay was evaluated in mt DNA samples pre-typed by Sanger sequencing. Sensitivity was tested by dilution series, described in detail elsewhere [4]. The reproducibility of the background signal (“false positive droplets”) was determined by calculation of mean and standard deviation of n= 50 non-template controls and n= 20 positive controls each for FAMor VIClabelled mt DNA samples. Residual white blood cells and their mt DNA content could interfere with the platelet quantification based on the mt DNA PCR. Samples prepared for mt DNA analysis were, therefore, tested for the presence of genomic DNA as an indicator for residual white blood cells as described before [4]. The absolute count of platelets was calculated from the readout of the droplet digital PCR system (copies/μL) as: concentration (copies/μL)/genome mass (pg) × dilution factor (PRP preparation; PCR assay). The cut-off value for quantification of platelets for all mt SNPs was set to 1000 platelets/μL based on dilution series. The third of three consecutive days with increasing platelet counts was defined as day of engraftment. A digital PCR for SNP detection was developed and validated to monitor the platelet engraftment in patients after allogeneic stem cell transplantation. DNA samples from 30 patients examined in our previous study for quantification of endogenous platelets with real-time PCR were used to validate the digital PCR assay [4]. The quantification of platelets in 150 samples was congruent in * Andrea Doescher doescher@bsd-nstob.de