First Measurement of a Long-Lived π

The adapted DIRAC experiment at the CERN PS accelerator observed for the first time long-lived hydrogenlike π^{+}π^{-} atoms, produced by protons hitting a beryllium target. A part of these atoms crossed the gap of 96 mm between the target and a 2.1 µm thick platinum foil, in which most of them dissociated. Analyzing the observed number of atomic pairs, n_{A}^{L}=436_{-61}^{+157}|_{tot}, the lifetime of the 2p state is found to be τ_{2p}=(0.45_{-0.30}^{+1.08}|_{tot})×10^{-11} s, not contradicting the corresponding QED 2p state lifetime τ_{2p}^{QED}=1.17×10^{-11} s. This lifetime value is three orders of magnitude larger than our previously measured value of the π^{+}π^{-} atom ground state lifetime τ=(3.15_{-0.26}^{+0.28}|_{tot})×10^{-15} s. Further studies of long-lived π^{+}π^{-} atoms will allow us to measure energy differences between p and s atomic states and so to discriminate between the isoscalar and isotensor ππ scattering lengths with the aim to check QCD predictions.

Observation of π

The observation of hydrogenlike πK atoms, consisting of π^{-}K^{+} or π^{+}K^{-} mesons, is presented. The atoms are produced by 24 GeV/c protons from the CERN PS accelerator, interacting with platinum or nickel foil targets. The breakup (ionization) of πK atoms in the same targets yields characteristic πK pairs, called "atomic pairs," with small relative momenta Q in the pair center-of-mass system. The upgraded DIRAC experiment observed 349±62 such atomic πK pairs, corresponding to a signal of 5.6 standard deviations. This is the first statistically significant observation of the strange dimesonic πK atom.

Experimental validation of a novel compact focusing scheme for future energy-frontier linear lepton colliders.

A novel scheme for the focusing of high-energy leptons in future linear colliders was proposed in 2001 [P. Raimondi and A. Seryi, Phys. Rev. Lett. 86, 3779 (2001)]. This scheme has many advantageous properties over previously studied focusing schemes, including being significantly shorter for a given energy and having a significantly better energy bandwidth. Experimental results from the ATF2 accelerator at KEK are presented that validate the operating principle of such a scheme by demonstrating the demagnification of a 1.3 GeV electron beam down to below 65 nm in height using an energy-scaled version of the compact focusing optics designed for the ILC collider.

Antitumor effects of the MIG and IP-10 genes transferred with poly [D,L-2,4-diaminobutyric acid] on murine neuroblastoma.

The number of tumor-infiltrating lymphocytes is known to be related to outcomes in patients with a variety of malignancies. Interferon (IFN) gamma-inducible protein-10 (IP-10) and monokine induced by IFNgamma (MIG) have chemotactic effects on activated T lymphocytes and natural killer (NK) cells. The aim of this study was to evaluate the antitumor effects of exogenous expression of the MIG and IP-10 genes delivered to solid tumors by poly [D,L-2,4-diaminobutyric acid] (PDBA). The murine MIG and IP-10 genes were transfected into mouse neuroblastoma cells with PDBA. MIG and IP-10 levels in supernatants of transfected cells were measured by enzyme-linked immunosorbent assay. The chemotactic activities of MIG and IP-10 in the supernatants of cell cultures were measured by chemotaxis assay. Tumors were injected in vivo with PDBA/pmMIGColon, two colonsIP-10 complexes to evaluate the effects of these genes on tumor volume and survival time of mice. Transfected PDBA/pmMIGColon, two colonsIP-10 complexes produced MIG and IP-10 protein in vitro. MIG and IP-10 proteins secreted into the culture medium showed chemotactic activity. MIG and IP-10 gene therapy with the PDBA system in vivo significantly inhibited tumor growth and prolonged survival time of mice. In conclusion, PDBA-mediated MIG and IP-10 gene therapy may be useful for treatment of solid tumors.

Preoperative transcatheter arterial chemoembolization reduces long-term survival rate after hepatic resection for resectable hepatocellular carcinoma.

AIMS: To study the effect of preoperative transcatheter arterial chemoembolization (TACE) on long-term survival after hepatic resection for hepatocellular carcinoma (HCC), we conducted a comparative analysis in 235 HCC patients who underwent hepatic resection with a curative intent. METHODS: We compared clinicopathologic background, mortality, and survival rates after hepatic resection between those who underwent preoperative TACE (n=109) and those who did not (n=126). RESULTS: One hundred and two patients in the TACE group (93.6%) received TACE only once. The mean interval between TACE and hepatic resection was 33.1days. Patients in the TACE group were younger than those in the non-TACE group, and liver cirrhosis and non-anatomical hepatic resection were more prevalent in this group. The 5-year overall survival rate after hepatic resection was significantly lower in the TACE group (28.6%) than in the non-TACE group (50.6%), especially in patients without cirrhosis or with stage I or II tumor. There was no difference between the two groups in mortality or disease-free survival after hepatic resection. Multivariate analysis showed preoperative TACE, preoperative aspartate aminotransferase elevation, and microscopic portal invasion to be independent risk factors for a poor outcome after hepatic resection. CONCLUSIONS: Preoperative TACE should be avoided for patients with resectable HCC, especially for those without cirrhosis or with an early stage tumor.

Irreversible morphological changes in leg bone following chronic gravitational unloading of growing rats.

Effects of gravitational unloading or loading on the growth and development of hindlimb bones were studied in rats. Male Wistar rats were hindlimb-unloaded or loaded at 2-G from the postnatal day 4 to month 3. The morphology and mineral content of tibia and fibula, as well as the mobility of ankle joints, were measured at the end of 3-month suspension or loading, and 1, 2, and 3 months after ambulation recovery. Growth-related increases of bone weight and mineral density were inhibited by unloading. But they were gradually recovered toward the control levels, even though they were still less than those in the age-matched controls after 3 months. None of the parameters were influenced by 2-G loading. However, here we report that chronic unloading causes abnormal morphological development in hindlimb bone of growing rats. Irreversible external bend of the shaft and rotation of the distal end of tibia, which limit the dorsiflexion of ankle joints, were induced following chronic gravitational unloading during developing period. It is also suggested that such phenomena are caused by the abnormal mechanical forces imposed by muscle utilization with altered patterns. The activity of ankle dorsiflexor was increased and that of plantarflexor was inhibited during unloading.

Compact permanent magnet H⁺ ECR ion source with pulse gas valve.

Compact H(+) ECR ion source using permanent magnets is under development. Switching the hydrogen gas flow in pulse operations can reduce the gas loads to vacuum evacuation systems. A specially designed piezo gas valve chops the gas flow quickly. A 6 GHz ECR ion source equipped with the piezo gas valve is tested. The gas flow was measured by a fast ion gauge and a few ms response time is obtained.

Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes.

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.

Membrane disorganization induced by perfringolysin O (theta-toxin) of Clostridium perfringens--effect of toxin binding and self-assembly on liposomes.

theta-Toxin (perfringolysin O) of Clostridium perfringens binds to membrane cholesterol with high (Kd approximately 10(-9) M) and low (Kd approximately 10(-7) M) affinities and causes membrane lysis of intact cells and liposomes. In order to understand the lytic process at the molecular level, the lysis of liposomes was investigated in comparison with that of intact cells. The toxin dose required to cause 50% lysis (RD50) of phosphatidylcholine/phosphatidylglycerol (82:18, mol/mol) liposomes containing 36-40 mol% cholesterol was 300-1400-times higher than the RD50 value for sheep or human erythrocytes when samples with the same cholesterol concentration were compared. However, the average number of toxin molecules bound per liposome vesicle at 50% lysis was estimated as 10-18 from the RD50 values, close to the number on erythrocytes at 50% lysis, suggesting that the number of toxin molecules adsorbed per vesicle is important for lysis. As to the toxin dose required for membrane lysis, no significant difference was observed between liposomes containing both high- and low-affinity toxin-binding sites and those containing only low-affinity sites, suggesting that theta-toxin molecules bound to low-affinity sites can assemble and cause membrane lysis as well as those bound to high-affinity sites. theta-Toxin assembles on liposomal membranes, as on erythrocytes, in a high-molecular-weight polymeric form as judged from sedimentation patterns in sucrose density-gradient centrifugation. The high-molecular-weight polymers were detected only under conditions where cell or liposome lysis occurred. At low toxin doses, slower sedimenting toxin oligomers and monomers were predominant on liposomal membranes. These results indicate that toxin assembly on membranes is essential for liposome lysis as it is for cell lysis and that assembly occurs on membranes without membrane proteins.

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.

Effect of perfringolysin O on the lateral diffusion constant of membrane proteins of hepatocytes as revealed by fluorescence recovery after photobleaching.

Perfringolysin O is a thiol-activated cytolytic exotoxin the primary receptor of which is the membrane cholesterol on the cell surface. The effect of perfringolysin O was tested in various hepatocyte preparations. (i) Smears of fresh liver exposed to a mild H2O2 (1.0 mM) injury for 10 min at 37 degrees C, develop a 'peroxide-induced autofluorescence' (PIAF) on the membrane proteins. PIAF is suitable for measuring the average lateral diffusion constant (D) of the membrane proteins by means of fluorescence recovery after photobleaching technique (FRAP). Incubation for 5 min with 600 or 2000 units/ml of the perfringolysin O resulted in a significant increase (32 and 46%, respectively) of D as compared to the controls of the same age group (13-14 months). Various tests like heat denaturation of cholesterol saturation of perfringolysin O before its application as well as thiol-activation of the smears with dithiothreitol revealed that the increase of D is a specific toxin effect due mot probably to the reaction of perfringolysin O with cholesterol. (ii) Isolated hepatocytes were exposed to perfringolysin O and their viability as well as the release of two cytosolic enzymes (lactate dehydrogenase and glutamic-pyruvic transaminase) were measured; 40-60 units/ml of perfringolysin O in 30 min reduced the viability of the hepatocytes to zero and caused a release of about 70% of both cytosolic enzymes. The significance of the results is discussed from the points of view of both the toxin-effect and the FRAP method.

A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol.

theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholesterol, we prepared a theta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erythrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by either flow cytometry or laser confocal microscopy. The fluorescence intensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither trypan blue nor propidium iodide, indicating that BC stained just the outer surface of the plasma membrane of vital cells. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensity of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVEC. This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BC theta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on the topology and distribution of cholesterol.

Adhesive surface determines raft composition in platelets adhered under flow.

Adhesion to von Willebrand factor (VWF) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) or rafts play a critical role in clustering of receptors that control these responses. Platelets adhered to VWF and collagen show CRDs concentrated in filopodia which contain both the VWF receptor glycoprotein (GP) Ibalpha and the collagen receptor GPVI. Biochemical analysis of CRDs shows a threefold enrichment of GPIbalpha (but not GPVI) in VWF-adhered platelets and a fourfold enrichment of GPVI (but not GPIbalpha) in collagen-adhered platelets. Depletion of cholesterol (i) leaves the initial adhesion unchanged, (ii) inhibits spreading on VWF and aggregate formation on collagen, (iii) leaves filopodia formation intact, and (iv) reduces the localization in filopodia of GPIbalpha but not of GPVI. These data show that the adhesive substrate determines the composition of CRDs, and that cholesterol is crucial for redistribution of GPIbalpha but not of GPVI.

The conserved undecapeptide shared by thiol-activated cytolysins is involved in membrane binding.

Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains.

The molecular mechanism that causes non-adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains--also known as rafts--play a crucial role in this process. We have used a non-cytolytic derivative of perfringolysin-O, a cholesterol binding cytolysin, that binds selectively to cholesterol-rich membrane domains, combined with confocal- and immunoelectron microscopy to visualize cholesterol-raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of alpha-granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion-dependent raft aggregation was accompanied by concentration of the tyrosine kinase c-Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c-Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low-density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP-induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co-cluster with the tyrosine kinase c-Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.

Assessment of donor fatty livers for liver transplantation.

AIM: The effect of fatty liver on graft survival, especially with reference to macrovesicular and microvesicular steatosis, is still uncertain. This preliminarily study was designed to create a noninvasive method for the quantification of the hepatic fat content in vivo and to establish provisional criteria for the assessment of fatty donor livers before liver transplantation among transplant surgeons, radiologists, and pathologists. METHODS AND MATERIALS: Different degrees of rat fatty liver model were established by feeding rats a diet deficient in choline and methionine for different periods of time. Computed tomography (CT) with test tubes containing variable percentages of fat equivalent substance were used to assess the severity of fatty change of the rat liver. This was then correlated with the histological classification, level of hepatic enzymes, and graft survival. RESULTS: Linear correlation between the fat volume fraction added to the test tubes and CT density were found. The process of producing a fatty liver via diet alteration peaked at week 3. At this time hepatic enzymes, radiological fat content, and posttransplantation survival were worse (P=0.013), compared with other time points. Radiological assessment of fatty liver correlated well with survival and serum glutamic oxaloacetic transaminase and glutamic pyruvate transaminase levels. CONCLUSION: Severe microvesicular steatosis does not influence recipient survival, however, macrovesicular steatosis affects graft survival. Caliber CT is a practical and simple method that allows an accurate noninvasive quantitative assessment of hepatic fatty infiltration. It has potential to be a useful parameter for the assessment of donor livers for clinical liver transplantation.

Crosslinked plasmalemmal cholesterol is sequestered to caveolae: analysis with a new cytochemical probe.

[symbol: see text]-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BC theta) to eliminate hemolyzing activity and was used as a cytochemical probe. In fixed cells, binding of BC theta was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BC theta and then with either fluorescein-avidin D or colloidal gold-streptavidin, the labeling in fine dots was distributed on the cell surface without local concentration as long as cells were kept on ice. When the temperature was raised to 37 C after treatment, the probe formed discrete large patches and became sequestered to caveolae. Binding of BC theta alone without the secondary reagents did not cause redistribution even at 37 C. Because the plasma membrane maintains integrity even after binding of BC theta, the probe can be used not only for cytochemical labelling of fixed cells but for pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that crosslinked cholesterol in the plasma membrane is sequestered to caveolae.

Colicin E3 induced cleavage of 16s rRNA of isolated 30S ribosomal subunits.

Contrary to the previous findings we found that a specific cleavage of 16S rRNA occurred when protein A, the active component of colicin E3, was interacted with isolated 30S ribosomal subunits at high concentrations. Necessary experiments to disprove the possibility of artefacts are described.

Protein synthesis in a cell-free system from an extreme thermophile. Effects of preincubation in the cold on polyuridylic acid-dependent polyphenylalanine synthesis at high temperature.

1. It was found that preincubation of the reaction mixture in the cold enhanced polyuridylic acid-directed polyphenylalanine synthesis by a cell-free extract of Thermus thermophilus HB8 at high temperature. 2. The effect of preincubation was most marked at 10-25 degrees in the presence of 20 mM Mg2+. Preincubation at 65 degrees failed to stimulate the incorporation. 3. The presence of phenylalanyl-tRNA, polyuridylic acid, and ribosomes was essential for preincubation in the cold to be effective. 4. A ternary complex of amino acyl-tRNA, polyuridylic acid, and a ribosome formed at low temperature was isolated by CPG-10 column chromatography; the isolated complex initiated polyphenylalanine synthesis effectively at high temperature. 5. The amount of the ternary complex formed depends on the preincubation time and the concentration of Mg2+. Since the amount of the complex correlated positively to the rate of polyphenylalanine synthesis at high temperature, the effectiveness of preincubation in the cold is presumably due to the formation of the ternary complex of phenylalanyl-tRNA, polyuridylic acid, and a ribosome.

Contribution of tryptophan residues to the structural changes in perfringolysin O during interaction with liposomal membranes.

Perfringolysin O (theta-toxin) is a cholesterol-binding and pore-forming toxin that shares with other thiol-activated cytolysins a highly conserved sequence, ECTGLAWEWWR (residues 430-440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of the toxin, we evaluated the contribution of each Trp to the toxin conformation during its interaction with liposomal membranes. Circular dichroism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp438, and Trp439 make large contributions to near-UV CD spectra. Quenching the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 and probably Trp436, but not Trp439, contributes to toxin insertion into the liposomal membrane. Near-UV CD spectra of the membrane-associated mutant toxins indicated that both Trp438 and Trp439 are required for the CD peak shift from 292 to 300 nm, a signal related to theta-toxin oligomerization and/or pore formation, suggesting a conformational change around Trp438 and Trp439 in these processes.