[Protein-dysregulation in human and murine myeloproliferative neoplasms]. / Protein-Dysregulationen in humanen und murinen myeloproliferativen Neoplasien.

In this work different types of dysregulation of signaling proteins in the context of myeloproliferative neoplasms are examined. In this heterogeneous disease group, uncontrolled cell proliferation plays a crucial role for the initiation of tumorigenesis, which Robert Weinberg described as a "hallmark" for the development of cancer. Protein dysregulation in form of overexpression of GAB2, a protein involved in formation of the CML-pathognomonic BCR/ABL-translocation complex, results in an enhanced disease phenotype in a Bcr/Abl-positive mouse model and disease acceleration is associated with a change of the subcellular localization of GAB2 in human blasts in CML-bone marrow biopsies. Furthermore, analyses of a mouse model show that a protein dysregulation caused by a distinct translocation (Tel-Syk) leads to the formation of a specific and morphologically very characteristic phenotype in the bone marrow of diseased mice. Moreover, results were presented which show that in certain subgroups of Myeloproliferative Neoplasms the protein NFE2, which is initially known only as a translocating factor, is apparently regulated by altering its subcellular localization. The difference in the subcellular localization of NFE2 in erythroid bone marrow cells is so clear between Essential Thrombocythemia and Primary Myelofibrosis that quantitative NFE2 immunohistochemistry can be used as an ancillary tool to diagnostically discriminate these two entities in an early stage.

Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation.

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in Eµ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eµ-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.

[Differential diagnosis of myeloproliferative neoplasms. Quantitative NF-E2 immunohistochemistry for differentiating between essential thrombocythemia and primary myelofibrosis]. / Differenzialdiagnose myelproliferativer Neoplasien. Quantitative NF-E2-Immunhistochemie zur Unterscheidung zwischen essenzieller Thrombozythämie und primärer Myelofibrose.

BACKGROUND: Besides essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) the myeloproliferative neoplasms (MPN) defined by the World Health Organization (WHO) comprise the entity of unclassifiable MPNs (MPN, U). The exact differential diagnosis of the specific MPN entities can be challenging particularly at early stages of the diseases. So far, pathologists have had to rely only on histomorphological evaluation of bone marrow biopsies in combination with laboratory data because helpful ancillary tests are not yet available. Even molecular tests, such as JAK2 mutation analysis are not helpful particularly in the differential diagnosis of ET and PMF because both entities are associated with the V617F mutation in 50 % of the cases. Recently overexpression of the transcription factor NF-E2 in MPN was described. MATERIALS AND METHODS: A collective of samples consisting of 163 bone marrow biopsies including 139 MPN cases was stained immunohistochemically for NF-E2 and analyzed regarding the subcellular localization of NF-E2 in erythroid progenitor cells. The results were compared between the MPN entities as well as the controls and statistical analyses were conducted. RESULTS AND DISCUSSION: This study showed that NF-E2 immunohistochemistry and analysis of the proportion of nuclear positive erythroblasts of all erythroid precursor cells can help to distinguish between ET and PMF even in early stages of the diseases. An MPN, U case showing a proportion of more than 20 % nuclear positive erythroblasts can be classified as a PMF with 92 % accuracy.

Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors.

Grb2-associated binder 2 (Gab2) serves as a critical amplifier in the signaling network of Bcr-Abl, the driver of chronic myeloid leukemia (CML). Despite the success of tyrosine kinase inhibitors (TKIs) in CML treatment, TKI resistance, caused by mutations in Bcr-Abl or aberrant activity of its network partners, remains a clinical problem. Using inducible expression and knockdown systems, we analyzed the role of Gab2 in Bcr-Abl signaling in human CML cells, especially with respect to TKI sensitivity. We show for the first time that Gab2 signaling protects CML cells from various Bcr-Abl inhibitors (imatinib, nilotinib, dasatinib and GNF-2), whereas Gab2 knockdown or haploinsufficiency leads to increased TKI sensitivity. We dissected the underlying molecular mechanism using various Gab2 mutants and kinase inhibitors and identified the Shp2/Ras/ERK and the PI3K/AKT/mTOR axes as the two critical signaling pathways. Gab2-mediated TKI resistance was associated with persistent phosphorylation of Gab2 Y452, a PI3K recruitment site, and consistent with this finding, the protective effect of Gab2 was completely abolished by the combination of dasatinib with the dual PI3K/mTOR inhibitor NVP-BEZ235. The identification of Gab2 as a novel modulator of TKI sensitivity in CML suggests that Gab2 could be exploited as a biomarker and therapeutic target in TKI-resistant disease.

[Peripheral facial palsy after embolization of a dural arteriovenous fistula with Onyx®]. / Periphere Fazialisparese nach Onyx®-Embolisation einer duralen arteriovenösen Fistel.

Dural arteriovenous fistulas (DAVF) are intracranial vascular malformations which can cause severe complications, such as intracranial bleeding and neurological deficits. We report the case of a patient presenting with pulsatile tinnitus caused by a DAVF. The DAVF was endovascularly treated including transarterial embolization with the liquid embolic agent Onyx®. The fistula and tinnitus were cured successfully, however, the rare complication of facial nerve palsy due to reflux of Onyx® into the vasa nervorum occurred. This could be confirmed directly during surgery and by histological analysis.

[Pseudo-mesotheliomatous adenocarcinoma of the lung]. / Pseudomesotheliomatöses Adenokarzinom der Lunge.

Both pathologists and clinicians are challenged by the diagnosis of a particular variant of the peripheral adenocarcinoma with involvement of the pleura parietalis, the so-called pseudo-mesotheliomatous adenocarcinoma of the lung (PMAC), which is hard to differentiate from epithelioid mesothelioma on imaging and cytology, macroscopically as well as histologically. However, the exact diagnosis is not only crucial for the patient's therapy but also for insurance matters. Immunohistochemical evaluation represents a quick and a relatively cheap tool for which a few antibody panels have been proposed in recent years as being suitable to distinguish between these two entities. One of the positive markers for epithelioid mesothelioma most often suggested seems to be calretinin. We would like to report on a case of PMAC with the special feature of positive calretinin immunohistochemical staining. Using histochemistry and a few additional antibodies we were able to reliably characterize the tumor and provide the patient with appropriate therapy. This article gives a short overview of the possibilities available for distinguishing between these two entities in the context of a case report.

The putative glutathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase.

A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infinite V(max) and K(m) when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55 s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3) m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4 x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.

Probing the presumed catalytic triad of a selenium-containing peroxidase by mutational analysis.

Glutathione peroxidases (GPx) are characterized by a catalytically active selenium which forms the center of a strictly conserved triad composed of selenocysteine, glutamine, and tryptophan. In order to check the functional relevance of this structural peculiarity, six molecular mutants of phospholipid hydroperoxide glutathione peroxidase (PHGPx) were designed, isolated, and investigated kinetically. Replacement of the selenocysteine in position 46 by cysteine decreased k + 1, i.e., the reaction rate of reduced enzyme with hydroperoxide, by three orders of magnitude. The rate of regeneration of the reduced enzyme by glutathione (k' + 2) was similarly affected. Additional substitution of Gln81 or Trp136 by acid residues resulted in a further decrease of k + 1 by three orders of magnitude, whereas histidine or neutral residues in these positions proved to be less deleterious. The data support the hypothesis that the typical triad of selenocysteine, glutamine, and tryptophan is indeed a novel catalytic center in which the reactivity of selenium is optimized by hydrogen bonding provided by the adjacent glutamine and tryptophan residues.

Glutathione peroxidase revisited--simulation of the catalytic cycle by computer-assisted molecular modelling.

Glutathione peroxidase, the first example of selenoproteins identified in mammals, was subjected to force field calculations and molecular dynamics in order to enable a clearer comprehension of enzymatic selenium catalysis. Starting from the established X-ray structure of bovine GPX, all kinetically defined intermediates and enzyme substrate complexes were modelled. The models thus obtained support the hypothesis that the essential steps of the catalysis are three distinct redox changes of the active site selenium which, in the ground state, presents itself at the surface of selenoperoxidases as the center of a characteristic triad built by selenocysteine, glutamine and tryptophan. In GPX, four arginine residues and a lysine residue provide an electrostatic architecture which, in each reductive step, directs the donor substrate GSH towards the catalytic center in such a way that its sulfhydryl group must react with the selenium moiety. To this end, different equally efficient modes of substrate binding appear possible. The models are consistent with substrate specificity data, kinetic pattern and other functional characteristics of the enzyme. Comparison of molecular models of GPX with those of other members of the GPX superfamily reveals that the cosubstrate binding mechanisms are unique for the classical type of cytosolic glutathione peroxidases but cannot operate e. g. in plasma GPX and phospholipid hydroperoxide GPX. The structural differences between the selenoperoxidases, shown to be relevant to their specificities, are discussed in terms of functional diversification within the GPX superfamily.

Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx).

Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis. The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively. Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed. The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact.. Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact. and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.

The design of enzyme sensors based on the enzyme structure.

The size of some of the reported electron mediators for glucose oxidase compared with the available space to penetrate the active site, implies that electrons have to move along the protein structure. Theoretical and experimental evidence predicts that it is possible to have electron transfer at the required rate used in biosensors (200 to 1840 electrons s-1) for the distances in glucose oxidase (20 to 25 A). Use of the program "Pathways" (together with the knowledge of the enzyme structure) allowed us to find an electron pathway within the enzyme. This pathway has a maximum electron coupling between the active site and the surface of the enzyme. The pathway reaches the surface near functional groups that can be used for oriented immobilization of the enzyme. Experimental confirmation of this particular pathway has been attempted but it is still elusive.

Phospholipid-hydroperoxide glutathione peroxidase. Genomic DNA, cDNA, and deduced amino acid sequence.

The complete amino acid sequence of the selenoprotein phospholipid-hydroperoxide glutathione peroxidase (PHGPX) from pig heart has been deduced from the corresponding genomic DNA, the cDNA covering the coding region, and by sequencing the N terminus of the protein. The maximum length of the peptide chain derived from the cDNA amounts to 170 amino acid residues. By protein sequencing the N-terminal residues methionine and cysteine of the deduced sequence were found to be cleaved. The molecular mass of 19,671 Da obtained by laser desorption mass spectroscopy, however, significantly exceeds the mean molecular mass of 19,257.09 calculated for the sequence 3-170 of PHGPX, thus indicating posttranscriptional modification. In contrast to glutathione peroxidase (GPX) the coding area of the PHGPX gene is composed of seven exons. Only the amino acid sequences encoded by the third and fifth exon are highly homologous to GPX sequences. The amino acid residues selenocysteine, tryptophan, and glutamine forming the catalytic site in bovine GPX are conserved in homologous positions of PHGPX, whereas the arginine residues presumed to bind GSH in GPX are not. Gaps in the PHGPX sequence correspond to subunit interaction sites of the tetrameric GPX. The data suggest an identical catalytic mechanism of the selenoperoxidases, a less stringent substrate specificity of PHGPX, and explain the monomeric nature of PHGPX. As in other selenoproteins, the selenocysteine residue of PHGPX is encoded by UGA. The 3'-untranslated region (UTR) of the PHGPX shows a limited consensus with that of GPX and 5'-deiodinase, where it was shown to be responsible for the decoding of UGA as selenocysteine. The 3'-UTR of PHGPX can form a stem/loop as in other mammalian selenoprotein genes. The 5'-UTR and the first intron of the PHGPX gene contain a variety of putative regulatory elements indicating hormonal control.

A structural model of mono- and diacylglycerol lipase from Penicillium camembertii.

The amino acid sequence of lipase from Penicillium camembertii was aligned with Rhizomucor miehei lipase without permitting any deletion or insertion in the structurally conserved regions. This lipase was classified into the R. miehei lipase family, because 33% of the residues were identical and 18% of the exchanges were conserved. A graphic molecular model for P. camembertii lipase was built using information from the sequence and X-ray structure of R. miehei lipase. The primary specificity pocket in the model of P. camembertii lipase predicted a substrate preference for monoacylglycerols and diacylglycerols. The close region to reactive His259 in P. camembertii lipase, which located in the opposite shore to the helical lid that was predictable to move in the activated state, contributed to the decision of the unique substrate specificity.

Taxonomical classification of the guinea pig based on its Cu/Zn superoxide dismutase sequence.

Cu-Zn superoxide dismutase was purified from guinea pig (Cavia porcellus) liver up to electrophoretic homogeneity and its amino acid sequence was elucidated by automated Edman degradation of proteolytic fragments and mass spectrometry. The protein was classified as a typical mammalian cytosolic Cu-Zn superoxide dismutase by molecular mass, specific activity, amino acid sequence and N-terminal acetylation. A dendrogram constructed from previously known vertebrate cytosolic Cu-Zn superoxide dismutase sequences reflects the commonly accepted taxonomy and phylogenetic relationships of the species, whereas the guinea pig sequence is similarly remote form muriform rodents, lagomorphs, equiforms and primates. The data appear incompatible with the assumption that the Caviomorpha with the representative Cavia porcellus form a common phylogenetic clade with the muriform rodents but rather have to be considered a distinct order of mammals. The degree of similarity of the sequences further suggests that the mammalian clade diverged into rodents, primates, lagomorphs and caviomorphs at about the same time.

Low temperature X-ray investigation of structural distributions in myoglobin.

The results of X-ray structure analysis of metmyoglobin at 300 K, 185 K, 165 K, 115 K and 80 K are reported. The lattice vectors a and b decrease linearly with temperature while c shows non-linearity above 180 K, indicating some type of phase transition. Cooling does change the myoglobin structure but only within the structural distribution as determined by individual (chi 2)-values at room temperature. Two residues showed significant alternative positions for side-chains at higher temperatures while only one position is occupied at low temperatures. In the case of LEU 61 a jump between different positions of the side-chain reduces the potential barrier for the entrance of the O2 molecule to the heme pocket. The mean square displacements, (chi 2), of the individual residues decrease linearly with temperature in most cases, indicating a parabolic envelope for the potential responsible for motions. A separation of rotational and translational disorder of the entire molecule is discussed. Comparison with Mössbauer spectroscopy indicates that protein dynamics on a time scale faster than 10(-7) s is not simply a harmonic process. Extrapolation of the structural distributions to T = 0 K shows that a large zero point distribution of the myoglobin structure exists, thus proving that there is no absolute energy minimum for one well defined conformation.