Comparative study on mechanoluminescence of irradiated and non-irradiated ionic crystals.

Effect of surrounding gas on mechanoluminescence (ML) of non-irradiated KCl:Ca2+ and X-ray irradiated one has been investigated in order to clarify the ML due to dislocation movement. For both samples, the ML appears only in the narrow pressure range from 10 to 10(3) Pa with a peak at 133 Pa. Emission spectrum of ML for both samples in Ne gas has indicated a prominent peak at 580 nm that coincides with a strong line of Ne gas. Above results indicate that the observed ML is caused by electric discharge at the crystal surface. Discussion is also made on the effect of Ca2+ on both the ML intensity and the charges carried with dislocations.

Thermoluminescence of photostimulable materials after X irradiation below room temperature.

Thermoluminescence glow peaks in the temperature range 100 to 400 K are investigated for BaFX (X = Cl, Br) crystals after X irradiation at 100 K. A prominent glow peak of BaFCl around 210 K is found to be composed of a few recombination roots, that is, the peak corresponds to the recombination of hole trapped centres such as an O- centre and a dissociated Cl2- centre with the F (F-) centre and the O2--F(Cl-) pair defect. Another small glow peak around 270 K is likely to occur from thermal dissociation of the O2- -F(Cl-) pair defect. The main glow peak of BaFBr:O2- at 170 K may be attributed to a recombination of an O- centre with the F(Br-) centre.

LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.

The molecular mechanisms that regulate basal or background entry of divalent cations into mammalian cells are poorly understood. Here we describe the cloning and functional characterization of a Ca2+- and Mg2+-permeable divalent cation channel, LTRPC7 (nomenclature compatible with that proposed in ref. 1), a new member of the LTRPC family of putative ion channels. Targeted deletion of LTRPC7 in DT-40 B cells was lethal, indicating that LTRPC7 has a fundamental and nonredundant role in cellular physiology. Electrophysiological analysis of HEK-293 cells overexpressing recombinant LTRPC7 showed large currents regulated by millimolar levels of intracellular Mg.ATP and Mg.GTP with the permeation properties of a voltage-independent divalent cation influx pathway. Analysis of several cultured cell types demonstrated small magnesium-nucleotide-regulated metal ion currents (MagNuM) with regulation and permeation properties essentially identical to the large currents observed in cells expressing recombinant LTRPC7. Our data indicate that LTRPC7, by virtue of its sensitivity to physiological Mg.ATP levels, may be involved in a fundamental process that adjusts plasma membrane divalent cation fluxes according to the metabolic state of the cell.

Regulation of the phospholipase C-gamma2 pathway in B cells.

In B lymphocytes, a signaling complex that contributes to cell fate decisions is the B-cell antigen receptor (BCR), with different extents of receptor engagement leading to such outcomes as cell death, survival, or proliferation. Here, based upon the available genetic and biochemical data of the BCR signal components, we discuss several mechanisms by which BCR signals are propagated and modified, with specific emphasis on the phospholipase C (PLC)-gamma2-calcium pathway Gene-targeting experiments in DT40 chicken B cells highlighted the importance of the intracellular protein tyrosine kinases Syk and Btk in PLC-gamma2 activation. Until recently, the molecular mechanism underlying the double requirement for Syk and Btk in PLC-gamma2 activation remained unclear, but new data suggest that an adapter molecule, B-cell linker protein (alternatively named SLP-65 or BASH), phosphorylated by Syk, provides docking sites for Btk SH2 domain as well as PLC-gamma2 SH2 domains, thus bringing Btk into close proximity with PLC-gamma2. The activated Btk then phosphorylates PLC-gamma2, leading to its activation. The activated PLC-gamma2 converts phosphatidylinositol 4,5-bisphosphate into the second messenger inositol 1,4,5-trisphosphate (IP3), which in turn binds to IP3 receptors located on the endoplasmic reticulum (ER). Binding of IP3 to the IP3 receptors is essential for triggering a calcium release from the ER and subsequent entry of extracellular calcium. Balancing these activation signals in the PLC-gamma2-calcium pathway are the inhibitory receptors expressed on B cells, FcyRII and paired immunoglobin-like receptor (PIR)-B. Although both FcyRII and PIR-B inhibits the BCR-mediated [Ca2+]i increase, the inhibitory mechanisms of these receptors are distinct. The FcyRII-mediated inhibitory signal is dependent on lipid phosphatase SHIP, whereas the PIR-B requires redundant functions of protein phosphatases SHP-1 and SHP-2. Thus, PIR-B and FcgammaRII inhibit calcium signals by utilizing two distinct signaling molecules, thereby contributing to setting threshold levels for activation signals as well as terminating activation responses.

Involvement of LAT, Gads, and Grb2 in compartmentation of SLP-76 to the plasma membrane.

B cell linker protein (BLNK) and Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76) are adaptor proteins required for B cell receptor (BCR) and T cell receptor function, respectively. Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells. This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking. Supporting this idea, the BCR function was restored when a membrane-associated SLP-76 chimera was enforcedly localized to GEMs. Moreover, we demonstrate that addition of both linker for activation of T cells (LAT) and Grb2-related adaptor downstream of Shc (Gads) to SLP-76 allow SLP-76 to be recruited into GEMs, whereby the BCR function is reconstituted. The Gads function was able to be replaced by overexpression of Grb2. In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.

The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions.

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

[Studies on bovine leukemia virus envelope glycoprotein gp30 YXXL sequences in virus infection and fusogenic activity].

The bovine leukemia virus (BLV) envelope transmembrane protein (gp30) contains three YXXL sequences at its cytoplasmic tail. It is known that N-terminal two of these sequences participate in the induction of B cell activation when chimeric proteins in which cytoplasmic domain of CD8-alpha has been replaced with that of BLV gp30 are stimulated by anti-CD8-alpha antibody. In addition to such signal transduction activity, the two tyrosines in the YXXL sequences also appear to involve infection with high viral loads and their maintenance in the sheep experimentally infected with BLV. To analyze detailed biological relevance of these sequences in vitro, we constructed a full-length BLV-infectious molecular clone with two copies of long terminal repeats (LTRs), designated pBLV-IF, and then changed residues Y487, L490, Y498, L501 or Y487 plus Y498 in gp30 cytoplasmic tail to Ala by site-directed mutagenesis. Introduction of molecular clones of wild-type and mutants into COS-1 cells revealed that all mutated molecular clones synthesized matured envelope proteins and released virus particles into growth medium. Serial passages of transient transfectants with the molecular clones and cell-free inoculation resulted in the reduction of infectivity by mutation of Y498 and Y487 plus Y498. In viral penetration, but not specific virus-cell binding, mutations of Y498 and Y487 plus Y498 substantially reduced the potential. Mutations of Y487, Y498 and Y487 plus Y498 increased syncytium-forming potential with increasing expression of envelope proteins on cell surface. In contrast, mutations of L490 and L501 affected neither infectivity nor syncytium-formation. Altogether, our data indicate that the YXXL sequences play a critical role during virus penetration and fusogenesity in the life cycle of BLV and regulate surface expression of envelope proteins.

Transmission and propagation in cell culture of virus produced by cells transfected with an infectious molecular clone of bovine leukemia virus.

A full-length molecular clone of bovine leukemia virus (BLV) pBLV-IF with two copies of a long terminal repeat (LTR) was constructed from a previously isolated, covalently closed, circular DNA clone, pB6490, that has one copy of the LTR and the pX region split at an EcoRI site. This molecular clone directed the synthesis of viral proteins and the induction of syncytia in transiently transfected cells. In addition, virus particles were released into the culture medium. Serial passages of transient transfectants also resulted in propagation of BLV. After transfection of five cell lines with linearized pBLV-IF and a neomycin-resistance gene, BLV-producing transfectants were established in cell lines COS-1 and 23CLN that did not form syncytia upon expression of BLV. In HeLa and FLK cells, BLV produced by a stable COS-1 transfectant was transmitted by both cell-free and cell-to-cell infection. Thus, pBLV-IF encoded an infectious provirus that successfully induced primary and secondary infections. This study indicates that the infectious molecular clone and the virus-producing transfectants could be useful for further examination of the biological properties of BLV.

CD44H participates in the intrahepatic growth of murine colon 26 adenocarcinoma cells.

The purpose of this study was to determine if CD44, a metastasis-associated cell adhesion molecule, is involved in the hepatic colonization by murine colon 26 adenocarcinoma cells. Indirect membrane immunofluorescence and FACS analysis showed strong expressions of CD44 and integrin beta 1 on colon 26 cells. Injection of 1 x 10(5) colon 26 cells into the superior mesenteric vein of syngeneic BALB/c mice produced macroscopic hepatic nodules in 92% (22/24) of the mice 14 days after inoculation. When colon 26 cells were pretreated with an anti-CD44 monoclonal antibody (mAb), IM7, only 30% (3/10) of the mice produced minute nodules in the liver on day 14 (P < 0.001), though IM7 did not inhibit growth of the cells in vitro. Pretreatment of colon 26 cells with an anti-integrin beta 1 mAb did not significantly block the hepatic metastasis. Histologically, microcolonies of tumor cells were detected in all of the livers on day 14 including the IM7-pretreatment mice that were free of gross nodules. However, percentages of tumor-occupied areas in the liver were consistently lower in IM7-pretreatment mice than in control mice (0.82% vs. 5.0% on day 14; P < 0.005). Reverse transcription-polymerase chain reaction (RT-PCR) amplification of mRNA revealed that colon 26 cells and splenocytes only expressed the hematopoietic isoform of CD44 (CD44H), which had no insertion of variant exons, while normal colonocytes expressed possible variant isoforms. These data suggest that malignant transformation of murine colonic epithelium altered the expression pattern of CD44 isoforms and that CD44H participates in the intrahepatic growth of colon 26 cells.

A sustained increase in beta-adrenoceptors during long-term therapy with metoprolol and bisoprolol in patients with heart failure from idiopathic dilated cardiomyopathy.

Effects of long-term therapy with beta 1-selective antagonists (metoprolol, bisoprolol) on beta-adrenoceptors in lymphocytes of patients with idiopathic dilated cardiomyopathy (DCM) were examined. There was a significant reduction in the number of lymphocyte beta-adrenoceptors in patients with DCM compared to that in healthy volunteers, as demonstrated by a selective decrease in maximum number of binding sites (Bmax) for (-)-[125I]iodocyanopindolol (CYP). A therapy with metoprolol and bisoprolol in these patients caused a marked increase in lymphocyte beta-adrenoceptor density. The significant increase was observed from 2 or 3 months after the start of therapy with these drugs, and it was maintained during the therapy for 24 months. The left ventricular ejection fraction in patients with DCM was improved by the long-term therapy with metoprolol and bisoprolol, and this effect seems to be correlated with an observed enhancement of lymphocyte beta-adrenoceptors in the time course. Also, the increase in lymphocyte beta-adrenoceptors appears to be correlated with a gradual amelioration in circulating catecholamine levels by the long-term therapy with beta-adrenoceptor antagonists in patients with DCM. Thus, the present study suggests that beta-adrenoceptors in lymphocytes of patients with DCM are up-regulated by a long-term therapy with metoprolol and bisoprolol.

Study on brain interstitial fluid distribution and blood-brain barrier transport of baclofen in rats by microdialysis.

PURPOSE: This study was performed to examine the distribution in the brain interstitial fluid (ISF) and the blood-brain barrier (BBB) transport of baclofen in rats by a microdialysis technique. METHODS: Following an i.v. bolus administration and/or the constant i.v. infusion of baclofen to the microdialysis cannula-bearing anesthetized rats, the concentrations of baclofen in the hippocampal ISF, whole brain tissue, cerebrospinal fluid (CSF), and plasma were determined by high-performance liquid chromatography (HPLC). Data were kinetically analyzed to estimate the transport parameters, i.e., the influx clearance (CLin) from plasma to brain and the efflux rate constant (keff) from brain to plasma, and the steady-state volume of distribution in the brain (Vd). RESULTS: The concentrations of baclofen in ISF, whole brain tissue, and CSF at the pseudo-steady state were almost 30-fold lower than the plasma unbound concentration, suggesting the restricted distribution of baclofen in the brain. The estimated values of CLin and keff were 0.00157 +/- 0.00076 ml/min/g of brain and 0.0872 +/- 0.0252 min-1, respectively. The efflux clearance (CLout) calculated by multiplying keff by Vd (0.816 +/- 0.559 ml/g of brain) was 0.0712 +/- 0.0529 ml/min/g of brain, and it was significantly 40-fold greater than the CLin value and fully greater than the convective flow in ISF. Furthermore, no significant concentration gradient was observed between ISF and CSF. These results suggest that the CLout value mainly reflects the efflux clearance through the BBB. Additionally, the hippocampal ISF/plasma concentration ratio of baclofen was markedly increased by both systemic administration of probenecid and its direct instillation into ISF. CONCLUSIONS: The restricted distribution of baclofen in the brain ISF may be ascribed to the efficient efflux from the brain through the BBB which is regulated possibly by a probenecid-sensitive organic anion transport system.

Angiotensin II receptor subtypes in bovine and human ventricular myocardium.

Angiotensin II (AII) binding sites in bovine and human ventricular myocardium were characterized by a radioreceptor assay. The specific binding of [125I]AII to myocardial membranes appeared to be saturable, and it was of high affinity with apparent dissociation constants of 1.60 nM (bovine) and 1.09 nM (human). The Bmax values were 39.7 fmol/mg protein (bovine) and 6.07 fmol/mg protein (human). Both losartan (angiotensin type 1 receptor subtype selective antagonist) and PD123177 (the angiotensin type 2 receptor subtype selective antagonist) inhibited specific [125I]AII binding to bovine myocardium, and their Hill coefficients were less than unity. Nonlinear least-squares regression analysis has suggested the existence of two populations of [125I]AII binding sites that have high and low affinity for losartan or PD123177. The relative proportions of high- and low-affinity sites for losartan in bovine myocardium were 68% and 32%, and those for PD123177 were 33% and 67%, respectively. On the other hand, specific [125I]AII binding in human myocardium was inhibited by losartan with much lower affinity and also by PD123177 with higher affinity, compared with bovine myocardium. The Hill coefficients for both drugs were close to one. Dithiothreitol enhanced specific [125I]AII binding to bovine myocardium in the presence of losartan, but it reduced [125I]AII binding in the presence of PD123177. This agent markedly enhanced specific [125I]AII binding to human myocardium. Thus, it is possible that bovine myocardium has both angiotensin type 1 receptor and the angiotensin type 2 receptor subtypes of AII receptors whereas human myocardium has predominantly the angiotensin type 2 receptor subtype.