High-Brightness Continuous-Wave Electron Beams from Superconducting Radio-Frequency Photoemission Gun.

Continuous-wave photoinjectors operating at high accelerating gradients promise to revolutionize many areas of science and applications. They can establish the basis for a new generation of monochromatic x-ray free electron lasers, high-brightness hadron beams, or a new generation of microchip production. In this Letter we report on the record-performing superconducting rf electron gun with CsK_{2}Sb photocathode. The gun is generating high charge electron bunches (up to 10 nC/bunch) and low transverse emittances, while operating for months with a single photocathode. This achievement opens a new era in generating high-power beams with a very high average brightness.

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2.

BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. FINDINGS: Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.

Effects of escitalopram on plasma concentrations of aripiprazole and its active metabolite, dehydroaripiprazole, in Japanese patients.

INTRODUCTION: The effects of escitalopram (10 mg/d) coadministration on plasma concentrations of aripiprazole and its active metabolite, dehydroaripiprazole, were studied in 13 Japanese psychiatric patients and compared with those of paroxetine (10 mg/d) coadministration. METHODS: The patients had received 6-24 mg/d of aripiprazole for at least 2 weeks. Patients were randomly allocated to one of 2 treatment sequences: paroxetine-escitalopram (n=6) or escitalopram-paroxetine (n=7). Each sequence consisted of two 2-week phases. Plasma concentrations of aripiprazole and dehydroaripiprazole were measured using liquid chromatography with mass spectrometric detection. RESULTS: Plasma concentrations of aripiprazole and the sum of aripiprazole and dehydroaripiprazole during paroxetine coadministration were 1.7-fold (95% confidence intervals [CI], 1.3-2.1, p<0.001) and 1.5-fold (95% CI 1.2-1.9, p<0.01) higher than those values before the coadministration. These values were not influenced by escitalopram coadministration (1.3-fold, 95% CI 1.1-1.5 and 1.3-fold, 95% CI 1.0-1.5). Plasma dehydroaripiprazole concentrations remained constant during the study. CONCLUSION: The present study suggests that low doses of escitalopram can be safely coadministered with aripiprazole, at least from a pharmacokinetic point of view.

A predictive factor of insufficient liver regeneration after preoperative portal vein embolization.

BACKGROUND: Preoperative portal vein embolization (PVE) is performed to enhance the future remnant liver function (FRLF) and volume (FRLV). However, the volume of the nonembolized liver does not increase enough in some patients, which results in an insufficient FRLF. The aim of this study was to evaluate the predictors of insufficient FRLF after PVE for extended hepatectomy. METHODS: This retrospective study included 172 patients (107 patients with cholangiocarcinoma, 40 patients with metastatic liver cancer and 25 patients with hepatocellular carcinoma) who underwent PVE before extended hepatectomy. The total liver function was evaluated by measuring the indocyanine green plasma clearance rate (KICG). Computed tomography volumetry was conducted to evaluate the total liver volume and FRLV. The KICG of the future remnant liver (remK) was calculated using the following formula: KICG × FRLV/total liver volume. The safety margin for hepatectomy was set at remK after PVE (post-PVE remK) ≥ 0.05. RESULTS: One hundred and twenty-three patients with a post-PVE remK level of >0.05 underwent hepatectomy without postoperative liver failure [sufficient liver regeneration (SLR) group], and 9 patients with a post-PVE remK level of <0.05 did not due to insufficient FRLF [insufficient liver regeneration (ILR) group]. In the SLR group, the KICG values did not change after PVE (median, 0.144-0.146, p = 0.523); however, the %FRLV and remK increased significantly (35.0-44.3%, p < 0.001 and 0.0488-0.0610, p < 0001, respectively). In contrast, in the ILR group, the KICG values decreased significantly (0.128-0.108, p = 0.021) and the %FRLV increased marginally (27.4-32.6%, p = 0.051). As a result, the remK did not increase significantly (0.0351-0.0365, p = 0.213). A receiver operating characteristic curve demonstrated an remK value of 0.04 obtained before PVE (pre-PVE remK) to be the optimal cutoff point for defective liver regeneration. The univariate and multivariate analyses revealed that a pre-PVE remK value of <0.04 was a factor for ILR. It was also correlated with postoperative liver failure in the analysis of the patients who underwent hepatectomy. CONCLUSIONS: The patients in the ILR group did not achieve SLR after PVE due to a significant decrease in the KICG and an insufficient increase in %FRLV. A pre-PVE remK value of <0.04 is a useful predictor of insufficient regeneration of the nonembolized liver, even after PVE.

Echocardiographic evaluation of deformity and enlargement of the canine mitral valve annulus associated with myxomatous degenerative mitral valve disease.

INTRODUCTION/OBJECTIVES: Quantitative evaluation of the morphology of the mitral valve annulus (MVA) in dogs with myxomatous mitral valve disease (MMVD) may improve the techniques of mitral valve plasty. This study aimed to compare the MVA morphology on echocardiography in normal dogs and dogs with MMVD and to compare the echocardiographic and intraoperative measurements of the MVA in dogs with MMVD. ANIMALS, MATERIALS AND METHODS: The study population comprised 59 healthy dogs (control group) and 371 dogs with MMVD (MMVD group). The anterior-posterior diameter and transversal diameter (TD) of the MVA and the aortic annulus diameter were measured by echocardiography to calculate the mitral valve flattening ratio, mitral annulus area (MAA), mitral annulus circumference (MAC), contraction ratio of the MAA and aortic annulus area. In the MMVD group, the mitral annulus diameter (MAD) was macroscopically measured during mitral valve plasty. Areas and lengths were divided by the body surface area (BSA) and √BSA, respectively, for comparative analyses. RESULTS: The systolic and diastolic anterior-posterior diameter/√BSA, transversal diameter/√BSA, MAA/BSA converted to a natural logarithm (Ln(MAA/BSA)), and MAC/√BSA was significantly higher in the MMVD group than the control group, whereas flattening ratio values and contraction ratio of the MAA was significantly lower. Neither the aortic annulus diameter /√BSA nor the Ln(aortic annulus area/BSA) significantly differed between groups. In the MMVD group, diastolic MAC/√BSA and MAA/BSA correlated significantly with the MAD/√BSA. CONCLUSIONS: The MVA is larger and rounder in dogs with MMVD than controls. Two-dimensional echocardiographic measures of MAA and MAC correlate well with intraoperative measures of MAD.

Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp 70.

Despite extensive study since the early 1980s, the mechanism by which newly synthesized protein precursors are unfolded in the cytoplasm and targeted correctly to the mitochondrial surface prior to translocation through the mitochondrial membranes is understood poorly. Recently, an N-ethylmaleimide (NEM)-sensitive cytoplasmic factor called mitochondrial import stimulation factor (MSF), which catalyses the ATP-dependent unfolding of precursor proteins, was described. Unlike the more general chaperone proteins of the hsp70 families, MSF not only unfolds proteins but also targets the unfolded precursor proteins to the mitochondria. Here, Mihara and Omura summarize what is known about MSF and speculate on how it, and other cytoplasmic factors, may be involved in mitochondrial import.

Membrane topogenesis of a type I signal-anchor protein, mouse synaptotagmin II, on the endoplasmic reticulum.

Synaptotagmin II is a type I signal-anchor protein, in which the NH(2)-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

Characterization of the signal that directs Tom20 to the mitochondrial outer membrane.

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product.

The human retinoblastoma gene (RB1) encodes a protein (Rb) of 105 kilodaltons that can be phosphorylated. Analysis of Rb metabolism has shown that the protein has a half-life of more than 10 hours and is synthesized at all phases of the cell cycle. Newly synthesized Rb is not extensively phosphorylated (it is "underphosphorylated") in cells in the G0 and G1 phases but is phosphorylated at multiple sites at the G1/S boundary and in S phase. HL-60 cells that were induced to terminally differentiate by various chemicals lost their ability to phosphorylate newly synthesized Rb at multiple sites when cell growth was arrested. These findings suggest that underphosphorylated Rb may restrict cell proliferation.

Successful renal transplantation for a patient with pyoderma gangrenosum.

Pyoderma gangrenosum (PG), a rare skin disease of unknown etiology, forms intractable skin ulcers at surgical or traumatic sites. This case is a 40-year-old woman with PG who experienced end-stage renal disease due to type 1 diabetes mellitus. Arteriovenous fistula (AVF) creation and peritoneal dialysis introduction were considered to be difficult, because this patient had a history of developing intractable aseptic ulcers at surgical sites. Therefore, she continued hemodialysis via a temporary catheter. With frequent catheter exchange, there was stenosis of both the femoral veins and the internal jugular vein. Therefore, a hemodialysis catheter that could be used for the long term was inserted into the left jugular vein as a final site. To prevent the patient not being able to continue hemodialysis, we performed a kidney transplantation to save her life. We performed a blood type-compatible, living donor kidney transplantation after confirming the absence of active skin lesions. The 69-year-old donor was her mother. Induction immunotherapy started with tacrolimus, mycophenolate mofetil, steroids, and basiliximab. Intravenous pulses of methylprednisolone were performed to prevent ulceration of the surgical site on days 0-2 (500 mg/d). The postoperative course was excellent. After the operation, ulceration of the surgical site was never observed. The serum creatinine value was 0.87 mg/dL at 6 months. To our knowledge, renal transplantations for a patient with PG has not been previously reported.

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax.

The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway.

A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a comparison with neutralization of mouse TNFalpha.

BACKGROUND AND PURPOSE: The p38 kinase regulates the release of proinflammatory cytokines including tumour-necrosis factor-alpha (TNFalpha) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762-0, we investigated the therapeutic potential of p38 inhibition and compared this to anti-mouse (m)TNFalpha antibody treatment in murine collagen-induced arthritis (CIA). EXPERIMENTAL APPROACH: Pharmacological profiles of Org 48762-0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)-induced inflammation in mice. The effects of Org 48762-0 and of mTNFalpha-neutralization on established arthritis were examined in murine CIA. KEY RESULTS: Org 48762-0 potently inhibited p38alpha kinase with a high degree of kinase selectivity. In cellular assays, Org 48762-0 reduced LPS-induced TNFalpha release. Oral administration of Org 48762-0 in mice showed drug-like pharmacokinetic properties and inhibited LPS-induced cytokine production. These pharmacological characteristics of Org 48762-0 prompted a comparison of therapeutic efficacy with mTNFalpha-neutralization in CIA. Org 48762-0 and anti-mTNFalpha antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762-0 treatment only. Further, micro-computed tomographical and histopathological analyses confirmed the protective effects of Org 48762-0 on joint damage. CONCLUSIONS AND IMPLICATIONS: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFalpha produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.

Genetic and epigenetic alterations in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a clonal disorder characterized by dyshematopoiesis and high susceptibility to acute myeloid leukemia (AML). As patients with MDS have widely variable prognosis, we need to stratify them according to chromosomal abnormalities, genetic alterations, and epigenetic deregulations associated with progression to AML in order to treat these patients appropriately. Recently, evidence has been accumulating on the molecular mechanism underlying self-renewal of stem cells. Specifically, we have been focusing on Polycomb-group (PcG) genes, which play an important role in supporting self-renewal. There is emerging evidence indicating that the PcG complexes are indispensable for sustaining stem cell activity and cancer stem cells. We have reported that the expression of BMI1, a member of PcG, in hematopoietic stem cells or progenitor cells predicts the prognosis of patients with MDS and progression to acute leukemia. And recent genome-wide analyses showed that major transcriptional regulators governing development are under the regulation of PcG complexes. Thus PcG not only provides a molecular marker for monitoring disease progression of MDS, but also provides a clue for elucidating a molecular mechanism underlying the disease progression, which may help in the development of a new therapeutic strategy against MDS. Herein, we describe cytogenetic, genetic and molecular aberrations in MDS, focusing on epigenetic alterations through PcG.

Identification of potential regulatory elements for the transport of Emp24p.

To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxyl-terminal region of Emp24p. Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed. The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p. Mutational analyses revealed that the two carboxyl-terminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport. The acceleration was sequence specific, and the terminal valine appeared to be more important. The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export. Hence the LV residues are a novel anterograde transport signal. The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal. On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step. It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down. Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi. Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.

1,25-Dihydroxyvitamin D3-regulated expression of genes involved in human T-lymphocyte proliferation and differentiation.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited the secretion of gamma-interferon from human T-lymphocytes activated by the calcium ionophore, A23187, or phytohemagglutinin with or without 12-O-tetradecanoylphorbol-13-acetate. The agent also inhibited cell proliferation and interleukin 2 secretion by these cells. The inhibition of gamma-interferon secretion was time and dose dependent and partially abolished by the addition of exogenous human recombinant interleukin 2. To elucidate the molecular events by which 1,25-(OH)2D3 inhibits cell proliferation and lymphokine secretion, complementary DNA probes were used to follow the expression of genes involved in human T-lymphocyte proliferation and differentiation. 1,25-(OH)2D3 inhibited the expression of interleukin 2 and gamma-interferon messenger RNA in human lymphocytes activated by phytohemagglutinin and 12-O-tetradecanoylphorbol-13-acetate. It also inhibited the accumulation of c-myc protooncogene messenger RNA and, to a lesser extent, interleukin 2 receptor messenger RNA in these cells. However, it did not affect the expression of the HLA-DR gene. These results suggest that 1,25-(OH)2D3 selectively regulates T-lymphocyte activation-related genes at the level of messenger RNA.

Vascular endothelial growth factor inhibits apoptotic death in hematopoietic cells after exposure to chemotherapeutic drugs by inducing MCL1 acting as an antiapoptotic factor.

We reported previously that vascular endothelial growth factor (VEGF) inhibits the apoptotic death of hematopoietic cells that is induced by exposure to ionizing radiation (O. Katoh et al., Cancer Res., 55: 5687-5692, 1995). In this study, we show that VEGF also inhibits apoptotic cell death that is induced by exposure to the chemotherapeutic drugs etoposide and doxorubicin. To elucidate the molecular mechanisms underlying this inhibitory effect of VEGF, we examined expression levels of BCL2 family proteins in CMK86, a human leukemia cell line, after treatment with VEGF. Northern blotting and immunoblotting analyses revealed that the expression level of MCL1, a member of the BCL2 family, was increased by VEGF. Moreover, to examine the effects of MCL1 on apoptotic cell death induced by exposure to etoposide, we generated a clonal U937 myeloid leukemia cell line transfected with vectors that promoted the constitutive expression of MCL1. MCL1 decreased the caspase 3 activity induced by exposure to etoposide and increased the viability of the transfected cells after etoposide exposure. Therefore, MCL1 may be involved in the inhibitory effect of VEGF on apoptotic cell death.

Cooperative regulation of c-myc expression in differentiation of human promyelocytic leukemia induced by recombinant gamma-interferon and 1,25-dihydroxyvitamin D3.

The biological activity of recombinant human gamma-interferon (IFN-gamma) to induce the phenotypic differentiation of human promyelocytic leukemia cell line HL-60 and to regulate c-myc expression was evaluated. Treatment with IFN-gamma increased monocyte-associated cell surface antigens detected by monocyte-specific monoclonal antibodies in a dose- and time-dependent manner. These antigenic changes were accompanied by a functional differentiation, determined by the increase of phagocytic capability and superoxide generation. IFN-gamma was also found to suppress the growth of HL-60 cells and reduce expression of a c-myc oncogene. These phenotypic and morphological changes to macrophage-like cells induced by IFN-gamma were similar to those by 1,25-dihydroxyvitamin D3, whereas the plasma membrane antigenic changes were different. Moreover, the combination of IFN-gamma and suboptimal doses of 1,25-dihydroxyvitamin D3 have synergistic effects in augmenting mature monocyte specific antigens (Mo2, 63D3, OKM1, and OKM5). In the reduction of c-myc expression by these drugs, a cooperative effect was observed with the inhibition of transferrin receptor expression and cell growth. These results indicate that human recombinant IFN-gamma induces a monocyte phenotype in the HL-60 cells via a mechanism different from the action of 1,25-dihydroxyvitamin D3.