IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases.

Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL-5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL-5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein-tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.

Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice.

The cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk, formerly bpk or atk), is crucial for B cell development. Loss of kinase activity results in the human immunodeficiency, X-linked agammaglobulinemia, characterized by a failure to produce B cells. In the murine X-linked immunodeficiency (XID), B cells are present but respond abnormally to activating signals. The Btk gene, btk, was mapped to the xid region of the mouse X chromosome by interspecific backcross analysis. A single conserved residue within the amino terminal unique region of Btk was mutated in XID mice. This change in xid probably interferes with normal B cell signaling mediated by Btk protein interactions.

Role of Bruton's tyrosine kinase in immunodeficiency.

The genetic defect associated with human X-linked agammaglobulinemia and murine X-linked immunodeficiency was recently shown to result from lack of function of a new cytoplasmic tyrosine kinase, called Bruton's tyrosine kinase (Btk). The phenotypes associated with these immunodeficiencies indicate that Btk plays a critical role in B-lymphocyte development. The distinctive protein structure of Btk and preliminary functional studies suggest that Btk may act in a novel manner in a variety of signaling pathways.

Genetic variations associated with diabetic nephropathy and type II diabetes in a Japanese population.

Genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy and type II diabetes. To identify the genetic polymorphisms associated with diabetic nephropathy and type II diabetes, we performed a genome-wide association study using single-nucleotide polymorphisms as genetic markers. We also analyzed polymorphisms within the genes encoding for the renin-angiotensin system that were considered as candidate genes for diabetic nephropathy susceptibility and the transcription factor 7-like 2 (TCF7L2) as a candidate for type II diabetes, in a large cohort of a Japanese population. A genome-wide association study identified SLC12A3 and engulfment and cell motility 1 gene as the new candidates for diabetic nephropathy and transcription factor-activating protein 2beta as a novel susceptibility gene for type II diabetes; this observation was based on the significant association between the polymorphisms within the genes and the corresponding diseases (P<0.0001). Further, we discovered that the genes encoding the angiotensin-converting enzyme, angiotensinogen, and angiotensin II type I receptor have a significant combinational effect on conferring susceptibility to diabetic nephropathy. Furthermore, TCF7L2 that has been reported as a convincing susceptibility gene for type II diabetes in Caucasian populations was also shown to be associated with type II diabetes in a Japanese population. These genes could be considered as strong susceptibility genes for diabetic nephropathy and type II diabetes in the Japanese, although the new candidates that have been identified by genome-wide screening need to be examined in greater detail by several replication studies.

Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.

High-resolution Brillouin spectroscopy with angular dispersion-type Fabry-Perot interferometer and its application to a quartz crystal.

Although the multichannel Brillouin spectroscopy with an angular dispersion-type Fabry-Perot interferometer (ADFPI) becomes a powerful tool for quick measurements, its resolution and contrast are not enough for the study of single crystals. A highly sensitive multichannel detector enables the ADFPI to use a solid etalon with high reflectivity (99.5%); hence, the high resolution and the high contrast of a spectrum are achieved. The finesse, the inverse of the resolution, reaches 100 with a 10 mm diameter of aperture size. The highest finesse of 140 is obtained by using a smaller diameter of 2 mm. The accuracy is examined by the measurement of a quartz crystal. The improvement in the resolution and contrast enables investigations of weak attenuation in a quartz crystal. The elastic anomaly of the alpha-beta transition of a quartz crystal is clearly observed both in sound velocity and attenuation. From the elastic constant c(11), the critical parameter K=0.76 is determined.

Role of polar nanoregions with weak random fields in Pb-based perovskite ferroelectrics.

In relaxor ferroelectrics, the role of randomly orientated polar nanoregions (PNRs) with weak random fields (RFs) is one of the most puzzling issues of materials science. The relaxation time of polarization fluctuations of PNRs, which manifests themselves as a central peak (CP) in inelastic light scattering, is the important physical quantity to understand the dynamics of PNRs. Here, the angular and temperature dependences of depolarized and polarized CPs in 0.44Pb(Mg1/3Nb2/3)O3-0.56PbTiO3 single crystals with weak RFs have been studied by Raman and Brillouin scattering, respectively. The CPs observed in Raman scattering show the very clear angular dependence which is consistent with the local tetragonal symmetry. It is different from the well-known local rhombohedral symmetry with strong RFs for Pb(Mg1/3Nb2/3)O3. In Brillouin scattering, depolarized and polarized CPs show two relaxation processes corresponding to transverse and longitudinal fluctuations of PNRs. The remarkable slowing down towards the Curie temperature was observed for transverse fluctuations in local tetragonal symmetry.

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice.

AIM: Dysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D). METHODS: The colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKAy mice, using EPC colony-forming assay (EPC-CFA). RESULTS: T2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types. CONCLUSION: T2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.

A novel method for constructing gene-targeting vectors.

We developed a simple and rapid method for constructing knockout vectors using inverse-PCR (IPCR). The method consists of three steps: (i) digestion of a target bacterial artificial chromosome with several restriction enzymes (six-base cutters) followed by self-ligation; (ii) IPCR using circular DNAs as templates and two primers which are oriented in opposite directions; and (iii) cloning into a vector containing a positive selection marker, which results in a typical replacement knockout vector. We successfully targeted three mouse genes including the HPRT gene using this method. Compared with the conventional method, this method requires much less time (no more than 3 weeks). Notably, this method requires only small amounts of sequence information (several hundred base pairs such as is available from expressed sequence tags) and can be extended to a systematic mass production of targeting vectors applicable to many organisms, including yeast.

The impact of including corticosteroid in a periarticular injection for pain control after total knee arthroplasty: a double-blind randomised controlled trial.

UNLABELLED: There is conflicting evidence about the benefit of using corticosteroid in periarticular injections for pain relief after total knee arthroplasty (TKA). We carried out a double-blinded, randomised controlled trial to assess the efficacy of using corticosteroid in a periarticular injection to control pain after TKA. A total of 77 patients, 67 women and ten men, with a mean age of 74 years (47 to 88) who were about to undergo unilateral TKA were randomly assigned to have a periarticular injection with or without corticosteroid. The primary outcome was post-operative pain at rest during the first 24 hours after surgery, measured every two hours using a visual analogue pain scale score. The cumulative pain score was quantified using the area under the curve. The corticosteroid group had a significantly lower cumulative pain score than the no-corticosteroid group during the first 24 hours after surgery (mean area under the curve 139, 0 to 560, and 264, 0 to 1460; p = 0.024). The rate of complications, including surgical site infection, was not significantly different between the two groups up to one year post-operatively. The addition of corticosteroid to the periarticular injection significantly decreased early post-operative pain. Further studies are needed to confirm the safety of corticosteroid in periarticular injection. TAKE HOME MESSAGE: The use of corticosteroid in periarticular injection offered better pain relief during the initial 24 hours after TKA.

Fano resonance of Li-doped KTa(1-x)NbxO3 single crystals studied by Raman scattering.

The enhancement of functionality of perovskite ferroelectrics by local structure is one of current interests. By the Li-doping to KTa(1-x)NbxO3 (KTN), the large piezoelectric and electro-optic effects were reported. In order to give new insights into the mechanism of doping, the microscopic origin of the Fano resonance induced by the local structure was investigated in 5%Li-doped KTN single crystals by Raman scattering. The coupling between the continuum states and the transverse optical phonon near 196 cm(-1) (Slater mode) caused a Fano resonance. In the vicinity of the cubic-tetragonal phase transition temperature, TC-T = 31 °C, the almost disappearance of the Fano resonance and the remarkable change of the central peak (CP) intensity were observed upon heating. The local symmetry of the polar nanoregions (PNRs), which was responsible for the symmetry breaking in the cubic phase, was determined to E(x, y) symmetry by the angular dependence of Raman scattering. The electric field induced the significant change in the intensity of both CP and Fano resonance. From these experimental results, it is concluded that the origin of the Fano resonance in Li-doped KTN crystals is the coupling between polarization fluctuations of PNRs and the Slater mode, both belong to the E(x, y) symmetry.

Paired immunoglobulin-like receptor B (PIR-B) inhibits BCR-induced activation of Syk and Btk by SHP-1.

Coligation of paired immunoglobulin-like receptor B (PIR-B) with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SHP-1 and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B; however the molecular target(s) of these phosphatases remain elusive. Here we show that PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igalpha/Igbeta, Syk, Btk and phospholipase C (PLC)-gamma2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-gamma2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-gamma2. Furthermore, we define a requirement for Lyn in mediating tyrosine phosphorylation of PIR-B. Based on these results, we propose a model of PIR-B-mediated inhibitory signaling in which coligation of PIR-B and BCR results in phosphorylation of ITIMs by Lyn, subsequent recruitment of SHP-1, and a resulting inhibition of the BCR-induced inositol 1,4,5-trisphosphate generation by dephosphorylation of Syk and Btk.

Regulation of kinetic properties of GluR2 AMPA receptor channels by alternative splicing.

The four subunits of the AMPA-type glutamate receptor (GluR1-GluR4 or GluR-A-GluR-D) exist in two distinct forms, flip and flop, generated by alternative splicing of a 115 bp region. The GluR2 subunit plays a key role in determining the functional properties of the AMPA receptor channel. In this study, we examined the differences in kinetic properties between the flip and flop splice variants of the GluR2 subunit expressed in Xenopus oocytes using fast agonist application techniques. Glutamate was applied to outside-out patches from oocytes with piezo-driven double-barreled application pipettes. Because homomeric receptor channels composed of the edited form of GluR2 (GluR2R) produce no appreciable current responses, we expressed the unedited form of GluR2 (GluR2Q) in oocytes, which produced large current responses sufficient for analysis of the kinetic properties. The time constant for desensitization during application of 1 mM glutamate was 5.89 +/- 0. 17 msec (n = 50) in flip and 1.18 +/- 0.05 msec (n = 37) in flop. The deactivation time constant was 0.62 +/- 0.06 msec (n = 10) in flip and 0.54 +/- 0.05 msec (n = 10) in flop. The steady-state nondesensitizing current was 6.8 +/- 0.4% (n = 53) of the peak current in flip, whereas it was almost negligible in flop, being only 1.1 +/- 0.1% (n = 36). The slower desensitization kinetics and larger steady-state current responses in the flip variant were also observed in heteromeric receptors assembled from GluR2Q/GluR2R. Thus, desensitization occurred much more prominently in the flop variant in the recombinant GluR2 receptor channels.

Purification and characterization of nuclear scaffold proteins which bind to a highly repetitive bent DNA from rat liver.

Our previous work (Hibino et al. (1992) Biochem. Biophys. Res. Commun. 184, 853-858) has shown that the binding affinities of a highly repetitive DNA component for rat nuclear scaffold proteins, P123 and P130, depend on the degree of sequence-directed bending of the helix axis. In the present experiment, these proteins have been purified and finally isolated by DNA-Sepharose column chromatography. The pI values of P123 and P130 were 7.2 and 8.1, respectively. The southwestern blotting revealed that a highly repetitive bent DNA (370-bp XmmI fragment) from rat liver binds readily to the isolated proteins under a hypotonic condition (50 mM NaCl) and that the level of the binding affinity for each protein was lowered with increasing NaCl concentration. The sedimentation analysis predicted that direct interaction between the XmnI fragment and P123 or P130 results in the formation of a complex which consists of two of the fragments and one molecule of the protein, alternatively, one of the fragment and three molecules of the proteins. Distamycin A, an antibiotic which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding of the fragment to P123 or P130, whereas neither removal of the bend nor binding inhibition was observed with chromomycin A3, an antibiotic specific for GC-rich sites in DNA. These results imply that AT-rich regions in a highly repetitive DNA component cause bending of the helix axis to be recognized by some of nuclear scaffold proteins.

Continuing V gamma 2 to J gamma 2 rearrangements of murine T cell receptor gamma genes in a B-committed immature cell line.

In SPL2-1-2, a murine B-committed immature cell line transformed with a temperature-sensitive mutant of Abelson virus, T cell receptor (TCR) gamma gene rearrangements, together with IgH gene rearrangements, were induced during culture at a non-permissive temperature (39 degrees C). During 11-12 months of culture, TCR gamma gene rearrangements occurred in all cells. In contrast to TCR gamma genes, neither TCR beta or TCR delta were detected even after 11-12 months of culture at a non-permissive temperature. The majority of the TCR gamma gene rearrangements observed here were V gamma 2 to J gamma 2 joinings and the remaining rearrangements were J gamma 1-linked. V gamma 1 to J gamma 4 and V gamma 3 to J gamma 3 joinings were not detected. Approximately 70% of cells with TCR gamma gene rearrangements produced normal-sized transcripts from the rearranged TCR gamma genes. Cloning and sequencing analysis of a cDNA from the transcripts demonstrated that the whole structure of the cDNA was similar to that of T-lineage cells. These results showed that TCR gene rearrangements were restricted to the gamma genes and that V gamma 2 to J gamma 2 joinings occurred preferentially in this B-committed immature cell line. Furthermore, TCR gamma gene rearrangements also occurred in intracytoplasmic mu-chain producing cells. This indicated that the existence of intracytoplasmic mu-chains did not prevent TCR gamma gene rearrangements, although the existence of mu-chains is known to inhibit further productive IgH gene rearrangements, (allelic exclusion). These results should provide many implications for the mechanism of TCR gene rearrangements, especially that of cross-lineage rearrangements.

Nucleotide deletion of T cell receptor V gamma 2 and J gamma 2 coding sequences at V gamma 2-J gamma 2 junctions in immature B cell lines.

Immature B cell lines transformed with a temperature-sensitive mutant of Abelson murine leukaemia virus undergo preferentially V gamma 2 to J gamma 2 joinings during culture at a non-permissive temperature (39 degrees C). Here we examined nucleotide addition and deletion at the V gamma 2-J gamma 2 junctions in these V gamma 2 to J gamma 2 joinings, in comparison with the V gamma 2-J gamma 2 junctional sequences reported previously in T cells. Forty-eight V gamma 2-J gamma 2 junctions were PCR-amplified and sequenced. Only three of 48 V gamma 2-J gamma 2 junctions had nucleotide addition. The average nucleotide deletion of V gamma 2 coding sequences at the V gamma 2-J gamma 2 junctions was 6.9 nucleotides in immature B cells and 5.1 nucleotides in T cells (p less than 0.05). Also, the average nucleotide deletion of J gamma 2 coding sequences at the V gamma 2-J gamma 2 junctions was 3.1 nucleotides in immature B cells and 1.9 nucleotides in T cells (p less than 0.01). The average of total number of the deleted nucleotides at the V gamma 2-J gamma 2 junctions was 10.0 nucleotides in immature B cells and 7.0 nucleotides in T cells (p less than 0.01). No correlation was found between the extent of the nucleotide deletion of the V gamma 2 coding sequence and that of the J gamma 2 coding sequence at each V gamma 2-J gamma 2 junction in immature B and T cells. These results demonstrated that nucleotide deletion at the V gamma 2-J gamma 2 junctions was significantly wider in immature B cells than in T cells.

Bruton tyrosine kinase gene mutations in Turkish patients with presumed X-linked agammaglobulinemia.

X-linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B-cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800-1821del, 1843-1847del, 1288-1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA.

Autocrine regulation of cell proliferation by estradiol and hydroxytamoxifen of transformed mouse Leydig cells in serum-free culture.

We have previously reported that the cloned cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor shows an estrogen-dependent enhancement of cell proliferation in medium supplemented with charcoal-dextran-stripped fetal bovine serum. To avoid the involvement of unknown factors present in the serum in the pathway for estrogen-dependent cell growth, the present study was designed to establish a serum-free culture system to which growth factors could be added. To this end, we subcloned B-1 cells from the parental tumor cell line. The proliferation of B-1 cells was markedly stimulated by the addition of 10(-11)-10(-8) M estradiol into the serum-free medium [Eagle's Minimum Essential Medium-Ham's F-12 (1:1, vol/vol) containing 0.2% (wt/vol) BSA]. Epidermal growth factor (0.1-50 ng/ml) or insulin (0.1-50 micrograms/ml) alone or in combination with 10(-8) M estradiol did not affect the proliferation rate of B-1 cells. In contrast, a greater than 10-fold molar excess of 4-hydroxytamoxifen blocked estradiol-induced cell proliferation, while 4-hydroxytamoxifen alone failed to show a stimulatory effect on cell multiplication. Additionally, the conditioned medium collected from estradiol-stimulated cells was found to contain a growth-promoting factor(s) whose activity was not antagonized by 4-hydroxytamoxifen. Nonstimulated cells secreted a significant but low level of the growth-promoting factor. Finally, B-1 cells were found to be estrogen dependent for cell proliferation in BALB/c mice. Their growth was markedly inhibited by the administration of tamoxifen to the host mice. These results indicate that the serum-free culture system presented here is suitable for studying the autocrine mechanisms of cell growth regulated by estrogens as well as triphenylethylene compounds.

The nucleotide and deduced amino acid sequences of porcine liver proline- beta-naphthylamidase. Evidence for the identity with carboxylesterase.

A cDNA clone for porcine liver proline-beta-naphthylamidase was isolated and sequenced. The deduced amino acid sequence of 567 residues was highly homologous with those of carboxylesterases (EC 3.1.1.1) previously reported for other species. In addition, proline-beta-naphthylamidase purified from porcine liver was shown to have strong activity towards p-nitrophenylacetate, a representative substrate for carboxylesterases. These results suggest that proline-beta-naphthylamidase is identical with carboxylesterase.

Functional and possible physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-1-derived macrophages.

Acetyl LDL (modified low-density lipoprotein), which is thought to be taken up through scavenger receptor A (SR-A), rapidly induced the appearance of phosphotyrosine proteins in monocytic THP-1-derived macrophages in vitro. The two alternative forms of Lyn (p53 and p56) were found to be tyrosine-phosphorylated within 30 s after the stimulation with acetyl LDL. The catalytic activity of Lyn measured by an in vitro kinase assay had also increased in acetyl LDL-stimulated THP-1-derived macrophages. Furthermore, Lyn could be co-immunoprecipitated with SR-A from the cell lysate. These observations suggest a functional and possible physical association of SR-A with Lyn in THP-1-derived macrophages, and also imply a possible involvement of Lyn in SR-A signal transduction.