CRTH2-dependent, STAT6-independent induction of cedar pollen dermatitis.

BACKGROUND: Airborne contact dermatitis to cedar pollen is a recently identified disease that generally affects individuals with cedar pollinosis of the nasal and/or ocular symptoms, as well as some patients with atopic dermatitis. OBJECTIVE: To elucidate the pathological mechanisms of cedar pollen dermatitis. METHODS: We established a mouse model of cedar pollen dermatitis by epicutaneous sensitization with Japanese cedar pollen antigen (Ag). RESULTS: Histologically, there was marked dermal cellular infiltrate, including eosinophils and mast cells, with epidermal thickening. The induction of dermatitis was accompanied by production of cedar pollen-specific IgE. In the lesional skin, IL-13, IL-18, eotaxin/chemokine (C-C motif) ligand (CCL) 11, regulated upon activation, normal T cell expressed and secreted/CCL5, macrophage-derived chemokine/CCL22 and thymus and activation-regulated chemokine/CCL17, but not IL-4 and IFN-gamma, were produced. Mast cell-deficient WBB6F1-W/W(v) mice failed to develop cedar pollen dermatitis, although regional lymph node cells proliferated in response to Cryptomeria japonica (Cry j) 1 and Cry j2 Ags in vitro. Surprisingly, the induction of dermatitis was independent of STAT6/IgE. In contrast, mice deficient in CRTH2, a receptor for prostaglandin D2 (PGD2), showed diminished inflammation. Consistent with this, ramatroban, a CRTH2 antagonist, significantly inhibited inflammatory cell infiltration. CONCLUSION: These data suggest that PGD2-CRTH2 signalling contributes to inflammation in cedar pollen dermatitis, and unlike cedar pollinosis of the nasal mucosa, STAT6 is not a therapeutic target for treatment.

[Successful off-pump coronary artery bypass for a patient with aplastic anemia].

We report a 61-year-old man with aplastic anemia who underwent successful off-pump coronary artery bypass (OPCAB) after being admitted for angina pectoris. Coronary angiography showed severe stenosis of the left main coronary artery. Preoperative WBC was 2,200/microl, neutrophil 704/microl, Hb 8.1g/dl, and PLT 16,000/microl. We conducted OPCAB on double vessels using left internal thoracic and radial artery grafts. Thirty units of platelets were transfused intraoperatively with little perioperaive hemorrhage. Because of high grade fever, we injected 150 microg granulocyte colony-stimulating factor (G-CSF) every 3 days postoperatively to prevent major infection. The combination of appropriate perioperative management and OPCAB yielded an effective result for a patient with severe hematological disorders causing pancytopenia.

A simple method using 31P-NMR spectroscopy for the study of protein phosphorylation.

Nonradioactive 31P-NMR spectroscopy has previously been used for the study of protein phosphorylations. However, the procedures does not seem to be easy for non-experts of this field, hence, this approach has not been widely used. We introduce here a simple protocol with 31P-NMR spectroscopy to study in vitro phosphorylation in receptor proteins. The effectiveness of this method was verified using synthetic peptides and recombinant proteins of the C-terminus of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor, whose phosphorylations are considered to have important roles in synaptic plasticity. We show that a decrease in the pH of the sample solution after the phosphorylation reaction is critical for the separation of the phosphorylation signals. In the analysis of the C-terminal portion of the GluR2 AMPA receptor, the phosphorylation sites of which had not hitherto been well clarified, we found the presence of at least three protein kinase C (PKC) phosphorylation sites. Furthermore, this method allows prediction of the origins of each of the phosphorylation peaks. Thus, the techniques we described here is useful for examination of protein phosphorylation and permits us to safely conduct repetitive experiments.

Regulation of phosphatidylcholine biosynthesis by mGluR1alpha expressed in human embryonic kidney 293 cells--A 31P-NMR study.

A recent report has demonstrated that inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release plays a crucial role in neurite growth. Here, using 31P-NMR, we examine whether activation of the metabotropic glutamate receptor 1 (mGluR1), which induces the production of IP3, could modulate phospholipid metabolism in human embryonic kidney 293 cells. mGluR1alpha- but not ionotropic glutamate receptor 1-expressing cells stimulated with glutamate exhibited a drastic reduction in the phosphorylcholine level, with corresponding increases in the level of phosphatidylcholine, a major phospholipid component. Quantitative analysis of cell growth revealed that mGluR1alpha-expressing cells cultured with 100microM glutamate were statistically significantly longer than the nontransfected cells. The effect was no longer observed following coincubation with a metabotropic glutamate receptor antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. These results suggest that mGluR1alpha activation triggers phosphatidylcholine biosynthesis and may contribute to neurite extension.

[The cause of polyurethane catheter cracking during constant infusion of etoposide (VP-16) injection].

We studied the cause of cracking of a clinically used polyurethane (PU) catheter during the constant infusion of etoposide (VP-16) injection (Lastet), administered without dilution to patients as a part of combination high-dose chemotherapy. After VP-16 injection was infused into the PU catheter at a constant infusion rate (30 ml/h) for 24 h, a decrease in the elasticity (36% of untreated) and on increase in the length of the catheter (3.7%) were observed. These changes were significantly higher than those treated with the control saline. The similar changes of the PU catheter were observed after treatment with a basal solution containing polyethylene glycol 400 (PEG 400), polysorbate 80 and ethanol, which is the vehicle of the VP-16 injection, and with ethanol alone. Moreover, obvious degeneration of the internal wall (occurrence of spots like melting) and cutting face (micro-cracking) of the catheter was observed with an electron microscope after treatment with the vehicle. On the other hand, the elasticity or extension of the PU catheter were not changed after treatment with saline or PEG 400. From these findings, it was suggested that the degeneration and subsequent cracking of the PU catheter during the infusion of VP-16 injection was caused by ethanol contained in its injection solution. No cracking or morphological changes of polyvinyl chloride (PVC) and silicone catheters were found after treatment with the vehicle solution. However, since it has been reported in previous reports that di(2-ethylhexyl)phthalate was leached from PVC bags, the high dose chemotherapy with the dilution-free VP-16 injection should be achieved safely and effectively using a silicon catheter, rather than the PU catheter.

Subcellular localization of the MEN, MLL/MEN and truncated MLL proteins expressed in leukemic cells carrying the t(11;19)(q23;p13.1) translocation.

The t(11;19)(q23;p13.1) translocation is exclusively associated with myeloid leukemias. Previously, we cloned several species of MLL/MEN chimeric cDNAs in a patient with myeloid leukemia carrying the t(11;19)(q23;p13.1) translocation. The MEN sequence directly followed the 5' region of MLL cDNA in some species and otherwise there presented an inserted sequence of 120 bp between the MLL and MEN sequences in others. Because the insertion sequence contained an in-frame termination codon, they coded only for the NH2-terminal part of MLL (truncated MLL). We also cloned the normal MEN cDNA in full-length with a cDNA library derived from K562 cells. We expressed the normal MEN, MLL/MEN chimeric and truncated MLL proteins in COS7 cells with the corresponding cDNAs and detected them with antibodies raised against the MEN and MLL peptides. Immunostaining and subcellular fractionation showed nuclear localization of all these proteins. These findings suggested that MLL/MEN chimeric cDNAs were actually translated into both MLL/MEN fusion and truncated MLL proteins and that they were localized in the nucleus of leukemic cells. Recently, Conaway et al. reported that MEN is an RNA polymerase II elongation factor. The leukemogenesis by the t(11;19)(q23;p13.1) translocation may have resulted from the alteration of transcription regulation induced by the MLL/MEN fusion protein and/or the truncated MLL protein.

Identification of PU.1 and Sp1 as essential transcriptional factors for the promoter activity of mouse tec gene.

Tec is a cytoplasmic protein-tyrosine kinase abundantly expressed in hematopoietic precursor cells. To investigate the mechanism regulating the expression of Tec molecule, we cloned and analysed 5' flanking region of mouse tec gene up to -2kb from the transcriptional initiation site. Luciferase assays using successive deletion mutants demonstrated that regions from -364 to -323 and from -122 to -63, which contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively, are important for the transcriptional activity. Gel-shift and supershift assays revealed that PU.1 and Sp1 bind to the these regions through their consensus binding motifs. In addition, introduction of mutations into these motifs resulted in marked decrease in the promoter activity. These results indicate that PU.1 and Sp1 are essential for the transcriptional activity of the tec promoter and suggest that the cooperation of PU.1 and Sp1 plays a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineages.

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Structurally altered Evi-1 protein generated in the 3q21q26 syndrome.

Overexpression of the Evi-1 gene appears to be a consistent feature of the 3q21q26 syndrome, an association of myeloid leukemias/myelodysplastic syndrome with a specific chromosomal aberration involving both 3q21 and 3q26, such as t(3;3)(q21;q26) or inv(3)(q21q26). The rearrangement in 3q26 has been reported to occur near the Evi-1 locus, implicating that it is the critical gene deregulated in the 3q21q26 syndrome. Here we present a structural abnormality of Evi-1 protein in a case with the 3q21q26 syndrome. In this case carrying typical inv(3)(q21q26), the 3q26 breakpoint is located within an intron of the Evi-1 gene, and resulted in overexpression of normally unexpressed, an aberrant form of Evi-1 protein, in which the C-terminal 44 amino acids of wild-type Evi-1 protein were truncated and replaced by five amino acids. The truncated Evi-1 protein is shown to increase AP1 activity when expressed in NIH3T3 cells as its wild-type counterpart. We also show that the origin of this peculiar type of rearrangement of the Evi-1 gene is not an artifact during establishment of the cell line, but is the event that occurred in the primary leukemic cells. Our results strongly support that the primary target for the 3q21q26 syndrome is the Evi-1 gene, and provide the first evidence that the structurally altered Evi-1 gene may be involved in the 3q21q26 syndrome.

An epidermal growth factor receptor-leukocyte tyrosine kinase chimeric receptor generates ligand-dependent growth signals through the Ras signaling pathway.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase that belongs to the insulin receptor family. LTK is mainly expressed in pre B cells and brain. Previously we cloned the full-length cDNA of human LTK, but no ligands have so far been identified, and hence, very little is known about the physiological role of LTK. To analyze the function of the LTK kinase, we constructed chimeric receptors composed of the extracellular domain of epidermal growth factor receptor and the transmembrane and the cytoplasmic domains of LTK and established cell lines that stably express these chimeric molecules. When cultured in medium containing EGF, growth of these cell lines was stimulated, and these fusion proteins became autophosphorylated and associated with Shc in vivo in a ligand-dependent manner. By treatment with EGF, Shc was associated with the Grb2/Ash-Sos complex. Our analyses demonstrate that LTK associates with Grb2/Ash through an internal adaptor, Shc, depending on a ligand stimulation. The LTK binding site for Shc was tyrosine 862 at the carboxyl-terminal domain and to a lesser extent tyrosine 485 at the juxtamembrane domain. Both of them are located in NP/AXY motif which is consistent with binding sites for Shc. These findings demonstrate that LTK can activate the Ras pathway in a ligand-dependent manner and that at least one of the functions of this kinase is involved in the cell growth.

Characterization of three erythropoietin (Epo)-binding proteins in various human Epo-responsive cell lines and in cells transfected with human Epo-receptor cDNA.

Molecular cloning of a cDNA for a mouse erythropoietin (Epo) receptor (EpoR) has facilitated the understanding of the structure of this receptor. However, there is, as yet, no explanation for the discrepancy between the protein recognized by specific antibodies against mouse EpoR and the unexpectedly larger species that can be cross-linked to labeled Epo. It is unclear whether the product of an unidentified gene is included in the EpoR complex. In the present study, we directly compared the cross-linking patterns for human EpoR that were endogenously expressed in three types of Epo-responsive cell, and that was artificially expressed in nonhematopoietic cells after transfection with cDNA for human EpoR. We observed that 85-kD and 105-kD proteins formed ligand-receptor complexes in all the human Epo-responsive cells and, furthermore, that the formation of a complex derived from the 70-kD protein was dependent on the level of expression of the cloned EpoR mRNA in these cells. By contrast, a prominent cross-linked band derived from the 70-kD protein and a weaker band derived from the 80- to 85-kD protein, but no band derived from the 105-kD protein, could be shown in the case of nonhematopoietic cells transfected with the human EpoR cDNA. These observations suggest that the cloned cDNA for human EpoR alone does not allow generation of the complete EpoR in nonhematopoietic cells and that the 105-kD Epo-binding protein may represent the product of an as yet unidentified gene that is expressed in hematopoietic cells.

A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner.

p47v-crk (v-Crk), a transforming gene product containing Src homology (SH)-2 and -3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, a 125-135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v-Crk, suggesting a direct signal mediator of v-Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs of v-Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v-src (v-Src), as well as by v-Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v-Src-associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk-associated substrate) is a common cellular target of phosphorylation signal via v-Crk and v-Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2-containing molecules.

The C-terminal SH3 domain of the mouse c-Crk protein negatively regulates tyrosine-phosphorylation of Crk associated p130 in rat 3Y1 cells.

We have isolated the mouse c-crk cDNA from a mouse liver cDNA library. It encodes 304 amino acids and consists mainly of SH2/SH3 regions. In Northern blot analysis, the mouse c-crk mRNA is expressed ubiquitously in every tissue and organ, suggesting that the c-Crk protein may be a common signal transducing molecule among tissues. In contrast to the v-Crk protein, which has a single SH3 domain, the c-Crk protein contains two, the more N-terminal SH3(1) domain and the C-terminal SH3(2) domain. To elucidate functions of these SH3 domains, we have constructed two c-crk mutants, B-crk and D-crk, which lack the SH3(2) and the SH3(1) domain, respectively. These mutants were expressed in rat 3Y1 cells, and examined for their transforming ability in terms of morphological phenotypes and for tyrosine phosphorylation profiles of cells expressing the mutant proteins. Morphological alteration and increased tyrosine phosphorylation of 130-140 kDa proteins, the major component of which is the Crk-associated p130, were observed in cells expressing B-Crk as well as those expressing v-Crk, but little in cells expressing c-Crk even at a similar level of expression. Although a highly tyrosine-phosphorylated form of the p130 was coimmunoprecipitated with c-Crk as well as B-Crk, the relative level of tyrosine phosphorylation of the p130, which is normalized to the amount of Crk protein immunoprecipitated, was 10 to 20 times higher in B-Crk-expressing cells than in c-Crk- or D-Crk-expressing cells. The present results indicate that the SH3(2) domain of mouse c-Crk protein negatively regulates tyrosine phosphorylation of the p130, and that lack of the SH3(2) domain in B-Crk and v-Crk may contribute, at least partly, to their morphological alteration or transforming ability through increasing tyrosine phosphorylation of the p130.

An anti-alpha-fetoprotein antibody-daunorubicin conjugate with a novel poly-L-glutamic acid derivative as intermediate drug carrier.

In studies on antitumor antibody-cytotoxic drug conjugates as potential antitumor agents with improved tumor specificity, daunorubicin [(DM) daunomycin] was conjugated with an affinity-purified horse antibody to rat alpha-fetoprotein (AFP) with a novel derivative of poly-L-glutamic acid (PLGA) as the intermediate drug carrier. A single masked thiol group first was introduced by PLGA, and the thiol group was generated from it after the linking of DM to PLGA at the carboxyl groups of PLGA. The thiol group was used selectively for binding PLGA-DM to antibody that had been modified so as to have the maleimide groups. The conjugates (DM:PLGA:immunoglobulin molar ratio, 19.6:2.8:1 or 11.8:1.1:1) were more potent than DM in in vitro cytotoxicity against the AFP-producing rat ascites hepatoma cell line AH66. In therapeutic experiments, the conjugates were more efficacious in prolonging the lives of AH66 hepatoma-bearing DONRYU rats than DM, antibody, a mixture of DM and antibody, or a conjugate similarly prepared with normal horse immunoglobulin.