[The Lymphoid Variant of HES (L-HES) as Differential Diagnose of Severe Asthma in Childhood]. / Ein T-Zell assoziiertes Hypereosinophilie-Syndrom (L-HES) als Differenzialdiagnose eines Asthma bronchiale.

Based on a case report an overview on the differential diagnostic considerations with respect to blood hypereosinophilia (HE) and hypereosinophilic syndromes (HES) in childhood is given. A 13-year-old boy was admitted for the clarification of an asthma. In the blood count an increased HE with 3 500/µl (30%) was found along with elevated total serum IgE and IL-5 level (2 000 IU/ml and 17 pg/ml). Lung function showed an obstruction (FEV1 38%). Radiologically the picture of bronchiectasis and mucus pluggine appeared. In the BAL a HE (76%) with raised IL-5 level was apparent. Histologically asthma was diagnosed with mucostasis, hypertrophy of the bronchial wall musculature and a lung HE. Differential-diagnostically an ABPA, a Churg-Strauss-Syndrome, a parasitosis, drug associated HE, allergies and malignant disease could be excluded. An aberrant T-cell clone in peripheral blood was detected by flow cytometry and T-cell receptor clonal rearrangements by PCR, leading to the diagnosis of a lymphoid variant of HES (L-HES). Failure to detect the FIP1L1-PDGFRA gene fusion and a normal bone marrow examination could exclude a neoplastic HES (HESN). After steroid initiation, prompt decrease of blood eosinophilia with resolution of symptoms was observed. Steroid discontinuation led to eosinophilia recurrence associated with disease symptoms. As steroid-sparing agent the immunosuppressive azathioprine was additionally given; steroid doses could be decreased and stopped in the course. This case demonstrated the range of HE evaluation in infancy. With asthma one should also consider the possibility of a L-HES.

[The Hypereosinophilic Syndromes in Childhood]. / Die Hypereosinophilie-Syndrome (HES) im Kindesalter.

The hypereosinophilic syndromes are rare disorders in childhood and require extensive differential diagnostic considerations. In the last years the earlier "idiopathic HES" called syndromes could be differentiated into molecular biologically, immunophenotypically and clinically more characterized heterogeneous diseases with high therapeutic and prognostic relevance. Nowadays the term HES summarizes diseases, which go hand in hand with a local or systemic hypereosinophilia (HE) connected with an organ damage. Depending on the cause of the HE one differentiates primary/neoplastic HES (HESN) from secondary/reactive HES (HESR). The latter develops reactively in connection with allergies, parasitosis, medications, neoplasia or a clonal increase of T-lymphocytes among others. With HESN the HE results from a clonal increase of eosinophilic granulocytes. While for some subgroups of the HESN (among others FIP1L1-PDGFRA fusion gene) the administration of a tyrosine kinase inhibitor is a new and effective therapy option, glucocorticoids still represent the medication of first choice for many not PDGFRA associated variants. Different immunomodulatory drugs or cytostatic agents are necessary to allow dose reduction of glucocorticoids. The promising therapy with anti-IL-5 antibodies is still not approved in infancy, could however become a treatment option in the future. Due to the present lack of knowledge about the HES in infancy the establishment of a register should be aimed for the treatment of HES in infancy.

Studies on argon collisions with smooth and rough tungsten surfaces.

The aim of this work is to investigate argon scattering behaviors on the smooth and rough tungsten surfaces. Current work deals with numerical simulation of nanoscale heat transfer process accompanying with rarefied gas-solid substrate interactions using molecular dynamics (MD) method. Taking into account that this method is very time consuming, MD simulation using CUDA capable Graphic Cards is implemented. The results found that imperfection of the surface significantly influences on gas atom's momentum change upon collision. However, the energy exchange rate remains unchanged regardless to the surface roughness. This finding is in contrast with the results in extant literatures. We believed the results found in this paper are important for both numerical and theoretical analyses of rarefied gas flow in micro- and nano-systems where the choice of boundary conditions significantly influences flow.

Rarefied gas flow through nanoscale tungsten channels.

The aim of this work is to investigate argon flow behaviors through the channels with three types of boundary conditions. Current work deals with numerical simulations of rarefied gas flow through nano-channels using the Molecular Dynamics method. Taking into account that this method is very time consuming, we implemented all the simulations using CUDA capable graphic cards. We found that the well-known and relatively simple Maxwell model of boundary conditions is able to reproduce gas flow through a tungsten channel with irregularities and roughness, while it results in a significant error in the case of a smooth metal surface. We further found that the flow rate through a relatively short channel correlates nonlinearly with the channel's length. This finding is in contrast with the results available in extant literature. Our results are important for both numerical and theoretical analyses of rarefied gas flow in micro- and nano-systems where the choice of boundary conditions significantly influences flow.

New statistical boundary conditions for argon-tungsten interactions.

In this study, scattering processes of argon beam impinging on tungsten surface are investigated numerically by applying molecular dynamics (MD) simulations. Energy transfer, momentum change, and scattering processes of argon gas atoms from W(110) surface are discussed. A new model of argon-tungsten (Ar-W) interaction is proposed. Based on the new proposed model, one can simplify the boundary conditions of this problem. The new boundary conditions are proved to be in line with previous experimental and theoretical results. This paper demonstrates how to proceed normalization and further conversion of the MD simulation results into boundary conditions. Application of the new proposed boundary conditions for Ar-W interactions provides a significant speedup of computations.

The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation.

As an oncoprotein, Eps8 participates in v-Src-induced cellular transformation. To delineate the underlying mechanism, we conducted a yeast two-hybrid screening and identified IRSp53S, a protein critical in cell mobilization, as one of the Eps8-binding partners from a human brain cDNA library. The association was mediated by the multiple proline-rich regions of Eps8 and the C-terminal SH3-WWB containing domains of IRSp53S. In this study, we observed that Eps8 modulated the expression of IRSp53 in v-Src-transformed cells (IV5), raising the question of whether Eps8/IRSp53 interaction was crucial in carcinogenesis. To address this issue, we generated IV5-expressing irsp53 siRNA cells. Attenuation of IRSp53 reduced cell proliferation of IV5 in culture dish and tumor formation in mice, which could be partly rescued by ectopically expressed human IRSp53S. In addition, IRSp53 knockdown impaired activity of phosphatidylinositol 3-kinase (as reflected by Pi-Ser473 AKT) and Stat3 (as reflected by Pi-Tyr705 Stat3), and reduced cyclin D1 expression that culminated to impede G(1)-phase cell-cycle progression. Ectopically expressed human IRSp53S, but not its Eps8-binding defective mutants (that is, Delta363 and PPPDA), rescued these defects and partly restored cell proliferation. Remarkably, through activation of Src, EGF increased the formation of Eps8/IRSp53 complex and Stat3 activation in HeLa cells. With these results, we show for the first time that IRSp53, through its interaction with Eps8, not only affects cell migration but also dictates cellular growth in cancer cells.

New tricyclic and tetracyclic pyranocoumarins with an unprecedented C-4 substituent. Structure elucidation of tamanolide, tamanolide D and tamanolide P from Calophyllum inophyllum of French Polynesia.

Three new pyranocoumarin derivatives, tamanolide (1), tamanolide D (2) and tamanolide P (3), were isolated from the almond seeds of Calophyllum inophyllum L. (Clusiaceae) grown in French Polynesia. These compounds, having an unprecedented C-4 isobutyl substituent, have been characterized as a new class of pyranocoumarins called tamanolides. Their structures were elucidated on the basis of 1D and 2D NMR techniques (COSY, NOESY, HSQC and HMBC) in association with MS (HR-ESI-MS) data analysis.

Analysis of trinucleotide repeats in different SCA loci in spinocerebellar ataxia patients and in normal population of Taiwan.

OBJECTIVE: To identify various subtypes of spinocerebellar ataxias (SCAs) among autosomal dominant cerebellar ataxia (ADCA) patients referred to our research center, SCA1, SCA2, SCA3/MJD (Machado-Joseph disease), SCA6, SCA7, SCA8 and SCA12 loci were assessed for expansion of trinucleotide repeats. PATIENTS AND METHODS: A total of 211 ADCA patients, including 202 patients with dominantly inherited ataxia from 81 Taiwanese families and nine patients with sporadic ataxia, were included in this study and subjected to polymerase chain reaction (PCR) analysis. The amplified products of all loci were analyzed on both 3% agarose gels and 6% denaturing urea-polyacrylamide gels. PCR-based Southern blots were also applied for the detection of SCA7 locus. RESULTS: The SCA1 mutation was detected in six affected individuals from one family (1.2%) with expanded alleles of 50-53 CAG repeats. Fourteen individuals from nine families (11%) had a CAG trinucleotide repeat expansion at the SCA2 locus, while affected SCA2 alleles have 34-49 CAG repeats. The SCA3/MJD CAG trinucleotide repeat expansion in 60 affected individuals from 26 families (32%) was expanded to 71-85 CAG repeats. As for the SCA7 locus, there were two affected individuals from one family (1.2%) possessed 41 and 100 CAG repeats, respectively. However, we did not detect expansion in the SCA6, SCA8 and SCA12 loci in any patient. CONCLUSIONS: The SCA3/MJD CAG expansion was the most frequent mutation among the SCA patients. The relative prevalence of SCA3/MJD in Taiwan was higher than that of SCA2, SCA1 and SCA7.

Distinct ligand binding properties of Mac-2-binding protein and mouse cyclophilin [correction of mousephilin] C-associated protein.

Human Mac-2-binding protein (Mac-2-BP) is a secreted glycoprotein that is widely expressed. It binds to the human macrophage-associated lectin Mac-2 and has been suggested to have a role in host defence. Mouse cyclophilin C-associated protein (mCyCAP) is also a secreted glycoprotein that binds with high affinity to cyclophilin C in the absence of the immunosuppressive drug cyclosporin A. The two proteins share a similar domain structure and considerable sequence identity, including a highly conserved scavenger receptor cysteine-rich domain, and both of them exert their function within the immune system. To elucidate whether these molecules are also functional homologues, we compared their ligand binding properties using cell lines which express Mac-2-BP or mCyCAP as well as transfected cell lines stably expressing mCyCAP or a mutant version lacking the scavenger domain. These experiments show that Mac-2-BP is unable to bind to either human or mouse cyclophilin C and thatmCyCAP cannot bind to Mac-2. The scavenger domain is not required for the interaction between mCyCAP and cyclophilin C. We conclude that these proteins may be part of a larger family of proteins of immunological importance in which closer functional homologues might exists.

A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdala.

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation.

Two isoforms of Eps8, p97Eps8 and p68Eps8, have been identified as the substrates for receptor tyrosine kinases. Our previous studies indicated that both tyrosyl phosphorylation and protein expression of Eps8 were elevated in v-Src transformed cells. In an attempt to examine the role played by p97Eps8 in tumorigenesis, we have first obtained cells overexpressing p97Eps8 and its pleckstrin homology (PH)-truncated variant. We then demonstrated that cells overexpressing p97Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. Furthermore, elevated serum-induced extracellular responsive kinase (ERK) activation was observed in p97Eps8 overexpressors. This enhanced ERK activation was sensitive to a MEK1 specific inhibitor PD98059 and was important for p97Eps8-mediated transformation, since transfection of vectors expressing dominant negative MEK1 and p97Eps8 abrogated focus formation by p97Eps8. In contrast, PH-truncated p97Eps8 failed to localize at the plasma membrane and that the truncated variant also did not elevate ERK activation and cellular transformation in response to serum stimulation. Our results thus indicated that: (i) the gene encoding p97Eps8 was an oncogene; (ii) p97Eps8-induced oncogenesis was partly mediated by ERK activation; and (iii) the PH domain of p97Eps8 was critical for its cellular localization, ERK activation and its ability to transform cells. Oncogene (2001) 20, 106 - 112.

Robust adaptive fuzzy-neural control of nonlinear dynamical systems using generalized projection update law and variable structure controller.

In this paper, a robust adaptive fuzzy-neural control scheme for nonlinear dynamical systems is proposed to attenuate the effects caused by unmodeled dynamics, disturbance, and modeling errors. A generalized projection update law, which generalizes the projection algorithm modification and the switching-sigma adaptive law, is used to tune the adjustable parameters for preventing parameter drift and confining states of the system to the specified regions. Moreover, a variable structure control method is incorporated into the control law so that the derived controller is robust with respect to unmodeled dynamics, disturbances, and modeling errors. To demonstrate the effectiveness of the proposed method, several examples are illustrated in this paper.

Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src.

Two eps8 isoforms, p97eps8 and p68eps8, were previously identified as substrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine content in v-Src transformed cells (IV5) revealed that both isoforms were highly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97eps8 detected in cells coexpressing both p97eps8 and active Src relative to that in cells expressing p97eps8 alone supported our hypothesis. The existence of common phosphotryptic peptides between in vitro 32P-labeled p97eps8 and p68eps8 indicated that these two proteins shared the same Src-mediated sites. Further in vitro binding assays demonstrated that p68eps8 was the major eps8 isoforms that could be precipitated by bacterial fusion protein containing Src SH3. Interestingly, both p68eps8 and p97eps8 were preferentially expressed in v-Src transformed cells and the presence of p68eps8 appeared to depend on Src. Since p97eps8 has been implicated in mitogenesis and tumorigenesis, its readiness to be phosphorylated and induced by v-Src might attribute to v-Src-mediated transformation.

c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function.

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.

Hepatitis C virus core protein fused to hepatitis B virus core antigen for serological diagnosis of both hepatitis C and hepatitis B infections by ELISA.

The sequence encoding the truncated core protein (amino acids 1-98) of hepatitis C virus (HCc) was expressed in E. coli for production of HCc(1-98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS1PreS2HCc(1-98). The HCc(1-98) and HBcPreS1PreS2HCc(1-98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity. They induced antibodies against HBcAg and/or HCV core protein in rabbits and in mice. Moreover, HBcPreS1PreS2HCc(1-98) is more immunogenic than HCc(1-98) in terms of anti-HCc induction. An ELISA that employed recombinant HCV core antigens of either HCc(1-98) or HBcPreS1PreS2HCc(1-98) to detect anti-HCc and/or anti-HBc antibodies was developed. Evaluation of serum samples with different status of HBV and HCV infections suggested that HCc(1-98) might be suitable for the determination of antibodies against HCV core protein, while HBcPreS1PreS2HCc(1-98) might be of value to detect HCV and/or HBV infection in donated blood in HBV low-prevalence countries.

Vanadate-dependent FAK activation is accomplished by the sustained FAK Tyr-576/577 phosphorylation.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase implicated in cell-matrix interaction and integrin signaling. It is well established that Tyr-397 is the FAK autophosphorylation site and Tyr-407, -576/577, -861, and -925 are the sites on murine FAK that are mediated by Src family kinases. To study how FAK is regulated by tyrosine phosphatase(s), cells overexpressing chicken FAK are treated with sodium vanadate. Both the phosphotyrosine content and the enzymatic activity of FAK are increased in response to vanadate. Interestingly, sustained FAK Tyr-576/577 and -863 phosphorylations are detected in vanadate-treated FAK overexpressors and are dependent on FAK autophosphorylation. Further analysis of sodium vanadate-treated FAK overexpressors reveals that the enhanced FAK kinase activity parallels its elevated Tyr-576/577 phosphorylation. Thus, we conclude that Src-mediated FAK phosphorylation is regulated by a tyrosine phosphatase(s) and may be of physioligical significance.

Cloning of vascular adhesion protein 1 reveals a novel multifunctional adhesion molecule.

Vascular adhesion protein 1 (VAP-1) is a human endothelial sialoglycoprotein whose cell surface expression is induced under inflammatory conditions. It has been shown previously to participate in lymphocyte recirculation by mediating the binding of lymphocytes to peripheral lymph node vascular endothelial cells in an L-selectin-independent fashion. We report here that the VAP-1 cDNA encodes a type II transmembrane protein of 84.6 kD with a single transmembrane domain located at the NH2-terminal end of the molecule and six potential N-glycosylation sites in the extracellular domain. In vivo, the protein exists predominantly as a homodimer of 170-180 kD. Ax endothelial cells transfected with a VAP-1 cDNA express VAP-1 on their cell surface and bind lymphocytes, and the binding can be partially inhibited with anti-VAP-1 mAbs. VAP-1 has no similarity to any currently known adhesion molecules, but has significant identity to the copper-containing amine oxidase family and has a monoamine oxidase activity. We propose that VAP-1 is a novel type of adhesion molecule with dual function. With the appropriate glycosylation and in the correct inflammatory setting, its expression on the lumenal endothelial cell surface allows it to mediate lymphocyte adhesion and to function as an adhesion receptor involved in lymphocyte recirculation. Its primary function in other locations where it is expressed, such as smooth muscle, may depend on its inherent monoamine oxidase activity.

Coding joint formation in a cell-free V(D)J recombination system.

V(D)J recombination assembles the variable portion of antigen receptor genes in developing lymphocytes and is the only site-specific recombination reaction known in vertebrates. A cell-free system has been established that performs DNA cleavage, end processing, and joining to yield V(D)J coding joints that exhibit structural features similar to those formed in vivo. The reaction has the expected substrate, metal ion, and RAG protein requirements. The efficiency of coding joint formation is reduced dramatically by uncoupling the cleavage and joining portions of the reaction, indicating that a postcleavage coding end complex facilitates joining. By varying the reaction conditions, nucleotide loss from coding ends and heterogeneity of coding joints can be regulated. This cell-free system provides a novel tool for detailed mechanistic analyses of the end processing and joining steps of V(D)J recombination.

A mouse molecular mimic of human vascular adhesion protein-1.

Human vascular adhesion protein-1 (VAP-1) is an endothelial sialoglycoprotein which exists in forms of Mr 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-1 is functionally defined by an inhibitory mouse mAb 1B2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5B11) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb 1B2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the 1B2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCyCAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immunoglobulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1.