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.

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.

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.

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.

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.

Endosonographic diagnosis of pneumatosis cystoides intestinalis in infancy.

We report pneumatosis cystoides intestinalis in a 10-month-old girl who developed bloody diarrhea following chemotherapy for leukemia. The diagnosis was made only by colonic endoscopic ultrasonography, whereas the abdominal plain radiogram and computed tomography failed to elucidate the diagnosis. She was successfully treated with hyperbaric oxygen therapy. Wider application of endoscopic ultrasonography may lead to the more frequent detection of pneumatosis cystoides intestinalis, currently a rare disorder.

Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis.

BACKGROUND: X-linked agammaglobulinemia (XLA), caused by mutations in Bruton's tyrosine kinase (BTK ), is the most common form of inherited antibody deficiency. We previously reported that a flow cytometric evaluation of BTK expression in monocytes could easily detect XLA as well as its carrier. OBJECTIVE: Our purpose was to perform further flow cytometric analysis in additional XLA families in Japan. METHODS: In all, 106 hypogammaglobulinemic males (from 91 families) of various ages with a lack of mature B cells (<1%) were investigated. RESULTS: Flow cytometric assessment revealed the deficient BTK expression status in 78 families (93 patients), and mutations in BTK were identified in 76 of 78 families with presumed XLA. Of the patients with normal BTK expression, 2 showed missense mutations in which the normal amount of altered BTK transcript would cause the XLA phenotype. As many as 30% of these patients with XLA were clinically or genetically recognized beyond 5 years of age. Higher concentrations (>300 mg/dL) of serum IgG were evident in the cases diagnosed among adults, seemingly preventing severe infections. Fifty-seven of 70 mothers of patients with BTK deficiency were diagnosed as obligate carriers on the basis of a bimodal BTK expression pattern. Nine of the remaining 13 mothers showing nonmosaic BTK expression had no mutations in 2 alleles; surprisingly, the other 4 mothers had the mutated alleles. CONCLUSIONS: A diagnostic approach based on flow cytometric assessment for XLA should be initially considered in genetic investigation of antibody deficiencies, regardless of the patient's age.

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults.

BACKGROUND: X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood. METHODS: Flow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize a 27 year old male patient with mild hypogammaglobulinemia (IgG, 635 mg/dl; IgM, 11 mg/dl; IgA, <5 mg/dl). He had suffered from frequent pneumonia since age 25 but had no history of frequent infections in his childhood or in adolescence. Sequencing of the BTK cDNA obtained from an Epstein-Barr virus-transformed B lymphoblastoid cell line derived from the bone marrow of the patient was performed to confirm a genetic defect. RESULTS: Flow cytometric analysis of cytoplasmic BTK protein in peripheral monocytes indicated that the patient presents a rare case of adult-onset XLA and that his mother is an XLA carrier. Sequencing of the BTK gene revealed a deletion of AG in the codon for Glu605 (AGT), resulting in an aberrant stop codon that truncates the BTK protein in its kinase domain. CONCLUSIONS: This case suggests that some XLA cases may remain undiagnosed because they only show mild hypogammaglobulinemia and they lack repeated infections in childhood. Flow cytometric analysis is a powerful method to screen these patients.

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.

Characterization of mutations, including a novel regulatory defect in the first intron, in Bruton's tyrosine kinase gene from seven Korean X-linked agammaglobulinemia families.

In this report, we describe seven mutations, including a novel single base pair substitution in intron 1, of the Bruton's tyrosine kinase (Btk) gene found in 12 Korean patients with X-linked agammaglobulinemia. Various mutations, including three novel genetic alterations, were discovered using single-strand conformation polymorphism analysis and direct DNA sequencing. The effect of the intron 1 point mutation (intron 1 +5G-->A) was further evaluated using reporter constructs. Using luciferase assay experiments, we showed that the transcriptional activity of the mutant was significantly lower than in normal counterparts, indicating that the intronic mutation was functional. In addition, DNase I footprinting analysis showed that a single protected region spanning the position +3 to +15 bp hybridized with a mutant-specific probe, but not with a wild-type probe. EMSA indicated that a distinct nuclear protein has the ability to bind the mutant oligonucleotides to produce a new DNA-protein complex. We also observed decreased expression of Btk proteins in monocytes of patients having the point mutation in intron 1. Taken together with the functional analysis, our results strongly suggest the existence of a novel cis-acting element, which might be involved in the down-regulation of Btk gene transcription. Precise definition of the regulatory defect in the Btk intron 1 may provide valuable clues toward elucidating the pathogenesis of X-linked agammaglobulinemia.

Four tyrosine residues in phospholipase C-gamma 2, identified as Btk-dependent phosphorylation sites, are required for B cell antigen receptor-coupled calcium signaling.

Activation of phospholipase C-gamma2 (PLCgamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCgamma2.

Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X-linked agammaglobulinaemia and carrier females.

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk) and is characterized by an arrest of B-cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X-chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.

Maternal germinal mosaicism of X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by abnormalities in tyrosine kinase (BTK), and is characterized by a deficiency of peripheral blood B cells. We studied cytoplasmic expression of BTK protein and analyzed the BTK gene (BTK) in peripheral blood mononuclear cells from two siblings with XLA and additional family members. Cytoplasmic expression of BTK protein in monocytes was not detected in either patient with XLA. A single base deletion (C563) in BTK-exon 6, which encodes the TH domain, was identified in both XLA patients. However, normal cytoplasmic expression of BTK protein in monocytes was detected in their mother without any BTK mutation. These results strongly suggest germinal mosaicism in the mother.

BLNK mediates Syk-dependent Btk activation.

Btk is a critical molecule in B cell antigen receptor (BCR)-coupled signaling, and its activity is regulated by Lyn and Syk. Although the molecular mechanism of Lyn-dependent Btk activation has been investigated, that of Syk-dependent Btk activation has remained unidentified. We have demonstrated that BLNK mediates Syk-dependent Btk activation. In a reconstitution cell system, coexpression of BLNK allows Syk to phosphorylate Btk on its tyrosine 551, leading to the enhancement of Btk activity. This phosphorylation depends on the interaction of Btk and BLNK by means of the Btk-Src homology 2 domain. The existence of such an activation mechanism is supported by the observation that the BCR-induced Btk phosphorylation and activation are significantly reduced in BLNK-deficient B cells as well as in Syk-deficient B cells. Although previous observations have identified the function of BLNK as the linker that integrates the action of Btk and Syk into downstream effectors such as phospholipase Cgamma2, our present study indicates another function of BLNK that connects the activity of Syk to that of Btk.

Renal crisis due to intimal hyperplasia in a patient with mixed connective tissue disease (MCTD) accompanied by pulmonary hypertension.

We report the case of a young female patient with mixed connective tissue disease (MCTD). She had marked pulmonary hypertension (PH) without lung fibrosis. She developed renal crisis after delivery by caesarean section. Renal biopsy revealed severe renal intimal hyperplasia with mild glomerular changes. The combination of intravenous pulse high-dose corticosteroid and cyclophosphamide (CPA) infusion and subsequent corticosteroid oral administration rescued her from renal crisis. This suggests that the possibility of co-incident renal intimal hyperplasia should be considered in patients with MCTD accompanied by PH. In addition, there might be some clinical benefit in combining high-dose corticosteroid with CPA infusion in the treatment of renal crisis due to intimal hyperplasia in MCTD.

Expression of a system L neutral amino acid transporter at the blood-brain barrier.

Amino acid transport system L has been proposed to be one of the major nutrient transport systems at the blood-brain barrier. Using immunohistochemical analyses, a system L transporter LAT1 was shown to be expressed in the brain capillary endothelial cells in rats. Because LAT1 was coexpressed with 4F2 heavy chain which brings LAT1 to the plasma membrane, LAT1 is proposed to be functional in the plasma membrane of brain capillary endothelial cells. Both LAT1 and 4F2hc immunoreactivities were detected in a double line appearance surrounding endothelial cell nuclei, suggesting both proteins are present in the luminal and abluminal membranes. LAT1 is, thus, a blood-brain barrier system L transporter responsible for the permeation of aromatic or branched-chain amino acids and amino acid-related drugs such as L-DOPA.

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.

Genetic defect in human X-linked agammaglobulinemia impedes a maturational evolution of pro-B cells into a later stage of pre-B cells in the B-cell differentiation pathway.

Surrogate light chains (lambda 5/VpreB) are selectively expressed in early precursors of B cells. B-cell defects in X-linked agammaglobulinemia (XLA) are caused by mutations in the gene for Bruton's tyrosine kinase. To elucidate the nature of early B-lineage cells in bone marrow (BM), samples from 13 XLA patients and 24 healthy controls of different ages were comparatively analyzed using an antihuman VpreB monoclonal antibody. Expression of surrogate light (SL) and mu-heavy chains were examined after cell membrane permeabilization because they are mainly expressed in the cytoplasm of early B-lineage cells. A flow cytometric analysis of normal BM identified 5 discrete cell types of B cells: mu(-)SL(++) (pro-B [B-cell progenitor]), mu(low)SL(++) (pre-B1a), mu(low)SL(+) (pre-B1b), mu(low)SL(- )(pre-B2), and mu(high)SL(- )(B). The large cells, presumably in cycling states, were enriched in pre-B1a cells. The frequencies of B-lineage cells in BM were higher in young children, and declined with advancing age. In contrast, XLA showed a profound reduction in BM B-lineage cells. In XLA BM, an expansion of pro-B cells with some small pre-B1a cells was marked, but other cells were negligible. These observations illustrate a B-cell maturation defect in XLA as well as a normal human B-cell differentiation pathway. The results suggest that the genetic defect in XLA may impede the evolution of pro-B cells beyond the earlier pre-B stage into the later stage of pre-B cells in B-cell development. (Blood. 2000;96:610-617)