Effects of intravenous apolipoprotein A-I/phosphatidylcholine discs on paraoxonase and platelet-activating factor acetylhydrolase in human plasma and tissue fluid

We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.     

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

The ion motive force (IMF) across the cell membrane is one of the most important sources of biological energy in any cell. The IMF is utilized for many essential biological activities, such as ATP synthesis, solute transport, nutrient uptake, protein secretion, flagella-driven motility, and so on (1). The IMF is the sum of the electrical (Δψ) and chemical (ΔpI) potential differences of ions such as protons (H⁺) (the proton motive force [PMF]) and sodium ions (Na⁺) (the sodium motive force [SMF]) across the membrane and is defined by Eq. 1:IMF=Vm+kBTq ln[ion]in[ion]ex,[1]where V_m is Δψ; [ion]_in and [ion]_ex are the internal and external ion concentrations, respectively; k_B is Boltzmann’s constant; T is the absolute temperature (in kelvins); and q is the charge of the ion. The Δψ corresponds to the membrane voltage (2).The flagellum of the enteric bacterium Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella) is a supramolecular motility machine consisting of the basal body, which acts as a bidirectional rotary motor; the hook, which functions as a universal joint; and the filament, which works as a helical propeller. The Salmonella flagellar motor is powered by a PMF across the cytoplasmic membrane. The motor consists of a rotor and multiple stator units, each of which acts as a transmembrane proton channel complex. The stator unit converts the proton influx through the channel into the force for high-speed rotation of the long helical filament (3, 4).For construction of the hook and filament structures at the cell exterior, a specialized protein transporter utilizes the PMF to transport flagellar building blocks to the distal end of the growing flagellar structure. The flagellar protein transporter consists of a PMF-driven export gate complex made of five transmembrane proteins, FlhA, FlhB, FliP, FliQ, and FliR, and an ATPase ring complex consisting of three cytoplasmic proteins, FliH, FliI, and FliJ (SI Appendix, Fig. S1) (5, 6). These proteins are evolutionarily related to those of the virulence-associated type III secretion systems of pathogenic bacteria, which inject effector proteins into eukaryotic host cells for invasion (7). Furthermore, the entire structure of the ATPase ring complex is structurally similar to the cytoplasmic F₁ part of F_OF₁-ATP synthase, which utilizes the PMF for ATP synthesis (8–10).FliI forms a homo-hexamer that hydrolyzes ATP at an interface between neighboring FliI subunits (10–12). FliJ binds to the central pore of the FliI ring (9). ATP hydrolysis by the FliI ATPase not only activates the transmembrane export gate complex through an interaction between FliJ and the C-terminal cytoplasmic domain of FlhA (FlhA_C) (13, 14) but also opens the entrance gate of the polypeptide channel through an interaction between FliI and the C-terminal cytoplasmic domain of FlhB (FlhB_C) (15). As a result, the export gate complex becomes an active proton/protein antiporter that couples an inward-directed H⁺ flow with an outward-directed protein export (SI Appendix, Fig. S1) (16). When the cytoplasmic ATPase complex becomes nonfunctional, the FlgN chaperone activates the Na⁺-driven export engine of the export gate complex over a wide range of external pH, allowing the export gate complex to drive Na⁺-coupled protein export (17, 18). The transmembrane domain of FlhA (FlhA_TM) acts as a transmembrane ion channel for the transit of both H⁺ and Na⁺ across the cytoplasmic membrane (17).A chemical potential gradient of either H⁺ (ΔpH) or Na⁺ (ΔpNa) is required for efficient inward-directed translocation of H⁺ or Na⁺ when FliH and FliI are absent (13, 17). Although the Δψ component is critical for flagellar protein export by the wild-type export gate complex (19), it remains unknown when and how Δψ is used for the flagellar protein export process. To clarify this question, we used the Salmonella MMHI0117 [ΔfliH-fliI flhB(P28T)] strain (hereafter referred to as ΔHI B*; Table 1) (20), in which the export gate complex uses both Δψ and ΔpNa at different steps of the flagellar protein export process (13, 17). We show that an increase in Δψ generated by an upward shift of the external pH from 7.0 to 8.5 activates flagellar protein export by this mutant even in the absence of ΔpNa, suggesting the presence of a Δψ-dependent activation mechanism for proton-coupled protein secretion by the export gate complex. We also show that an increased Δψ facilitates efficient docking of FliJ to FlhA_C.Table 1.Summary for flagellar protein export properties of Salmonella strains used in this study

A case of drug-induced ductopenia resulting in fatal biliary cirrhosis

ABSTRACT— A 50-year-old woman suffered from a diffuse skin rash, high fever and jaundice immediately after a second injection of glutathion and Stronger Neo-minophagen C which contains glycyrrhizin. Liver biopsy performed 11 months after the onset showed mild spotty hepatocyte necrosis, marked cholestasis in parenchyma, and some lymphocyte infiltration in the portal area. Interlobular bile ducts had undergone vacuolar degeneration or were absent in some portal tracts. In her hospital course, unremitting jaundice persisted and biliary cirrhosis developed with signs of portal hypertension; she died from liver failure 26 months after the onset. A liver specimen at her death revealed that most of the interlobular bile ducts had vanished. Based on the clinical course and pathology, drug-induced ductopenia, possibly due to an adverse reaction to glycyrrhizin, is the most likely diagnosis. While drug-related biliary cirrhosis is rarely fatal, this case presented an unusually rapid course of fatal biliary cirrhosis.     

Nonoperative Removal of Bilateral Intrahepatic Biliary Stones by Endoscopic Electrohydraulic Lithotripsy

We report the successful treatment of bilateral intrahepatic biliary stones by endoscopic electrohydraulic lithotripsy (EHL). EHL is a useful procedure by which large stones can be fragmented easily and complete removal of stones can be attained.     

Diagnosing infection of extracardiac conduit in children

Summary Between August 1985 and July 1992, at our center, 142 Japanese children had an extracardiac conduit operation to reconstruct the right ventricular outflow tract. The study group consisted of 22 of these 142 children who had a persistent fever and whose serum was positive for acute-phase reactants after the operation. We present the diagnostic findings for 10 children with infection of an extracardiac conduit that had been placed to restore the continuity of the right ventricle-pulmonary artery. They were part of the group of 22 children who were followed over the past 7 years with blood cultures, echocardiography, and⁶⁷Ga imaging. All 10 developed a persistent fever and were seropositive for acute-phase reactants. Conduit infection was diagnosed in only 2 patients by the detection of vegetation on echocardiography and was diagnosed in 9 of the 10 patients by an abnormal⁶⁷Ga uptake in the area of the artificial vessels used to reconstruct the pulmonary artery. The present study compared the use of blood cultures, echocardiography, and⁶⁷Ga imaging in diagnosing an infection of the extracardiac conduit. The sensitivity of blood cultures in diagnosing an extracardiac conduit infection was 70% (7/10), and the specificity was 92% (1/12).⁶⁷Ga imaging showed a higher sensitivity than echocardiography in diagnosing infection of an extracardiac conduit.     

Long-term clinical effect of nilvadipine in patients with chronic heart failure: A double-blind placebo-controlled study

Summary The long-term effect of calcium channel blockers on chronic heart failure is disappointing, probably because of reflex sympathetic activation through arterial vasodilation. However, nilvadipine may be beneficial for treatment of chronic heart failure since this drug has minimal effects on sympathetic activation. In this study, the effects of 12-week administration of nilvadipine or placebo on symptoms of heart failure and cardiac function were investigated in 23 patients with mild-to-moderate chronic heart failure in a double-blind trial. The patients were randomly assigned to either a nilvadipine group (16 mg daily) or a placebo group. Intergroup comparisons did not show significant differences in any parameters. Serious adverse effects were not observed during the study. Thus, this study failed to show any beneficial effect of nilvadipine in the long-term treatment of patients with chronic heart failure. We conclude that the long-term administration of nilvadipine (16 mg daily) is neither effective nor harmful in the treatment of patients with chronic heart failure.Other members are listed in the appendix.     

RGS4 and GAIP are GTPase-activating proteins for Gqα and block activation of phospholipase Cβ by γ-thio-GTP-Gqα

RGS proteins constitute a newly appreciated and large group of negative regulators of G protein signaling. Four members of the RGS family act as GTPase-activating proteins (GAPs) with apparent specificity for members of the G_iα subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for G_qα, the G_α protein responsible for activation of phospholipase Cβ. Furthermore, these RGS proteins block activation of phospholipase Cβ by guanosine 5′-(3-O-thio)triphosphate-G_qα. GAP activity does not explain this effect, which apparently results from occlusion of the binding site on G_α for effector. Inhibitory effects of RGS proteins on G protein-mediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.     

Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration

Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KIT(WT)(WT) showed cell migration and Ca(2+) mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca(2+) mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca(2+) influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca(2+) influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca(2+) influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3'-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca(2+) mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca(2+) influx-Erk and the other is Y719-PI3K-Ca(2+) influx.