Down-regulation of the two-component system and cell-wall biosynthesis-related genes was associated with the reversion to daptomycin susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus.

Daptomycin (DAP) is widely used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. The emergence of DAP non-susceptible MRSA strains during therapy is a major concern in clinical settings. Recent studies revealed that MRSA spontaneously reverts to a subsequent methicillin-susceptible S. aureus (MSSA) strain. However, it is not clear whether DAP non-susceptible MRSA has the ability to revert to a susceptible strain. We obtained an MRSA strain pair, DAP non-susceptible strain and subsequent DAP susceptible strain, from a patient. To understand the underlying mechanism by which DAP non-susceptible MRSA reverts to a susceptible strain, we performed genetic and phenotypic analysis in the strain pair. Although whole-genome analysis revealed four missense mutations, including L826F in mprF, in both strains, the net cell-surface charge was similar between the DAP non-susceptible and susceptible strains. However, the thickness of the cell wall was higher in the DAP non-susceptible strain, which was decreased to the same level as the control after reversion to the DAP susceptible strain. Moreover, the non-susceptible strain showed higher mRNA expression of the two-component system (TCS), such as VraSR, yycG and GraS, with the up-regulated transcription levels of cell-wall biosynthesis-related genes. The expression levels of those genes were decreased after reversion to the susceptible strain. These results indicated that DAP non-susceptibility due to up-regulation of the TCS and cell-wall biosynthesis-related genes may be reversible by the discontinuation of DAP, leading to reversion to the DAP susceptible phenotype.

Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice.

AIMS/HYPOTHESIS: The pancreas and hypothalamus are critical for maintaining nutrient and energy homeostasis, and combined disorders in these organs account for the onset of the metabolic syndrome. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. The physiological role of ATF3 in the pancreas has been controversial, and its role in the hypothalamus remains unknown. To elucidate the roles of ATF3 in these organs, we generated pancreas- and hypothalamus-specific Atf3 knockout (PHT-Atf3-KO) mice in this study. METHODS: We crossed mice bearing floxed Atf3 alleles with Pdx1-cre mice, in which cre is specifically expressed in the pancreas and hypothalamus, and analysed metabolic variables, pancreatic morphology, food intake, energy expenditure and sympathetic activity in adipose tissue. We also used a hypothalamic cell line to investigate the molecular mechanism by which ATF3 regulates transcription of the gene encoding agouti-related protein (Agrp). RESULTS: Although PHT-Atf3-KO mice displayed better glucose tolerance, neither plasma glucagon nor insulin level was altered in these mice. However, these mice exhibited higher insulin sensitivity, which was accompanied by a leaner phenotype due to decreased food intake and increased energy expenditure. We also observed decreased hypothalamic Agrp expression in PHT-Atf3-KO mice. Importantly, an increase in ATF3 levels is induced by fasting or low glucose in the hypothalamus. We also showed that ATF3 interacts with forkhead box-containing protein, O subfamily 1 (FoxO1) on the Agrp promoter and activates Agrp transcription. CONCLUSIONS/INTERPRETATION: Our results suggest that ATF3 plays an important role in the control of glucose and energy metabolism by regulating Agrp.

Mesp2 initiates somite segmentation through the Notch signalling pathway.

The Notch-signalling pathway is important in establishing metameric pattern during somitogenesis. In mice, the lack of either of two molecules involved in the Notch-signalling pathway, Mesp2 or presenilin-1 (Ps1), results in contrasting phenotypes: caudalized versus rostralized vertebra. Here we adopt a genetic approach to analyse the molecular mechanism underlying the establishment of rostro-caudal polarity in somites. By focusing on the fact that expression of a Notch ligand, Dll1, is important for prefiguring somite identity, we found that Mesp2 initiates establishment of rostro-caudal polarity by controlling two Notch-signalling pathways. Initially, Mesp2 activates a Ps1-independent Notch-signalling cascade to suppress Dll1 expression and specify the rostral half of the somite. Ps1-mediated Notch-signalling is required to induce Dll1 expression in the caudal half of the somite. Therefore, Mesp2- and Ps1-dependent activation of Notch-signalling pathways might differentially regulate Dll1 expression, resulting in the establishment of the rostro-caudal polarity of somites.

[Resection of malignant fibrous histiocytoma through a combined thoracic and abdominal wall approach].

We report a case of resection of malignant fibrous histiocytoma (MFH) via combined thoracic and abdominal wall incision reconstructed using GORE DUALMESH. A 60-year-old woman underwent resection of a left lower chest wall tumor. Since the tumor infiltrated into the diaphragm, a part of the left diaphragm and left upper abdominal wall were resected together. The left chest was closed by suturing the diaphragm to the ribs. The resected area of the thoracic and abdominal wall was 12×12 cm and was reconstructed with GORE DUALMESH. She received adjuvant radiotherapy as the tumor cells were detected in the surgical margin of the diaphragm. The patient has remained well without signs of recurrence for 10 months after the operation.

Effect of an immunotoxin to folate receptor beta on bleomycin-induced experimental pulmonary fibrosis.

It has been suggested that alveolar and interstitial macrophages play a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) by producing proinflammatory and/or fibrogenic cytokines. We showed that inflammatory macrophages expressed folate receptor beta (FRbeta) while resident macrophages in normal tissues expressed no or low levels of FRbeta. In the present study, we examined the distribution of FRbeta-expressing macrophages in the lungs of patients with usual idiopathic pulmonary fibrosis (UIP) and mice with bleomycin-induced pulmonary fibrosis (PF) and tested whether the depletion of FRbeta-expressing macrophages could suppress bleomycin-induced PF in mice. Immunostaining with anti-human or -mouse FRbeta monoclonal antibody (mAb) revealed that FRbeta-expressing macrophages were present predominantly in fibrotic areas of the lungs of patients with UIP and mice with bleomycin-induced PF. Intranasal administration of a recombinant immunotoxin, consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRbeta mAb and truncated Pseudomonas exotoxin A, increased survival significantly and reduced levels of total hydroxyproline and fibrosis in bleomycin-induced PF. In immunohistochemical analysis, decreased numbers of tumour necrosis factor-alpha-, chemokines CCL2- and CCL12-producing cells were observed in the immunotoxin-treated group. These findings suggest a pathogenic role of FRbeta-expressing macrophages in IPF. Thus, targeting FRbeta-expressing macrophages may be a promising treatment of IPF.

mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse.

The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation-dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify mDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for mDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.

Defining the landscape of ATP-competitive inhibitor resistance residues in protein kinases.

Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.

Hif-2alpha mediates UV-induced apoptosis through a novel ATF3-dependent death pathway.

In this study, we describe a novel activating transcription factor 3 (ATF3)-dependent death pathway triggered by ultraviolet (UV) irradiation. We demonstrate that ATF3 contributes to UV-induced apoptosis through the regulation of hypoxia inducible factor (Hif)-2alpha expression, which in turn induces the expression of proapoptotic genes, such as Caspase7 or TRAIL (tumor necrosis factor (ligand) superfamily, member 10). Gain of function of Hif-2alpha as well as ATF3 is sufficient to trigger cell death, whereas loss of function of both proteins drastically inhibits UV-induced apoptosis. Repression of Hif-2alpha strongly impairs ATF3-mediated death, providing evidences that Hif-2alpha is the major death effector of ATF3. In addition, Hif-1alpha, already known as a proapoptotic gene, upon UV irradiation, is not able to compensate for the lack of Hif-2alpha expression, thereby confirming the major contribution of Hif-2alpha in UV-mediated cell death. We further demonstrate that this cascade of gene activation depends on p38 and c-Jun N-terminal kinase (JNK) activity. Impairment of such a pathway is likely to contribute to oncogenesis by promoting survival of cells that could accumulate severe chromosomal alterations.

[Aortitis syndrome with repeated restenoses of a drug eluting stent].

The patient was a 49-year-old female who developed acute myocardial infarction of the right coronary artery in August 2005. In a short period of time, the patient had restenosis repeatedly after percutaneous coronary intervention (PCI). Restenosis could not be prevented even with a drug eluting stent (DES), and thus, off-pump coronary artery bypass grafting (OPCAB) was performed. The diagnosis of aortitis syndrome was made due to protracted postoperative inflammation. Aortitis syndrome was determined to be the main cause of repeated restenosis. This case was a middle-aged female who had restenosis in a short period of time, and aortitis syndrome should have been included in the differential diagnosis. Although some positive results have been reported on DES placement for coronary lesions of aortitis syndrome, DES was completely ineffective in our patient. Further studies with more patients are necessary to examine the effectiveness of DES.

Induction of apoptosis and cellular senescence in mice lacking transcription elongation factor, Elongin A.

Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To gain more insight into the physiological functions of Elongin A, we generated Elongin A-deficient mice. Elongin A homozygous mutant (Elongin A(-/-)) embryos demonstrated a severely retarded development and died at between days 10.5 and 12.5 of gestation, most likely due to extensive apoptosis. Moreover, mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos exhibited not only increased apoptosis but also senescence-like growth defects accompanied by the activation of p38 MAPK and p53. Knockdown of Elongin A in MEFs by RNA interference also dramatically induced the senescent phenotype. A study using inhibitors of p38 MAPK and p53 and the generation of Elongin A-deficient mice with p53-null background suggests that both the p38 MAPK and p53 pathways are responsible for the induction of senescence-like phenotypes, whereas additional signaling pathways appear to be involved in the mediation of apoptosis in Elongin A(-/-) cells. Taken together, our results suggest that Elongin A is required for the transcription of genes essential for early embryonic development and downregulation of its activity is tightly associated with cellular senescence.

Diagnostics of a He(+) beam extracted from a compact magnetic bucket-type ion source.

Performance of a compact He(+) ion source was investigated before installing it to the experiment system developed for studying the efficiency of autodetachment neutralization from He(-) to He(0). Dependence of the extracted ion current upon the plasma parameters and that upon the extraction voltage indicated that the source performance was limited by space charge effect. The beam emittance was measured with a multislit system for various operation conditions. The experimentally determined emittance agreed well with the emittance obtained from ion trajectories calculated with input plasma parameters measured by a Langmuir probe. The beam current density of 60 mA/cm(2) and the emittance of less than 25pi mm mrad are expected to deliver enough current to the downstream of the He(-) neutralizer system to clarify factors affecting the final He(0) intensity.

Energy straggling of low-energy ion beam in a charge exchange cell for negative ion production.

Energy straggling in a charge exchange cell, which is frequently used for negative ion production, was studied experimentally and compared with the results of theoretical evaluation. The change of the energy spectrum of a He(+) beam due to charge exchange processes in argon gas was measured in the energy range of 2-6 keV. Energy straggling by multiple collisions is expressed by the energy loss formula due to inelastic and elastic processes. The impact parameter is related to the elastic scattering angle, and the geometry of the charge exchange cell and other components of the beam transportation system determines the maximum acceptable scattering angle. The energy spread was evaluated taking the integral limit over the impact parameter into consideration. The theoretical results showed good agreement with those of actual measurement.

A beam transport system for an intense He(-) beam source.

We have been developing a test stand for fast He(0) beam production. One of the major issues is how to transport effectively the He(+) and He(-) beams from which the He(0) beam is produced. The beam should be focused in two transverse focal points, the center of the charge exchange cell and the electrostatic accelerator. We studied the beam transport system and effect of space charge neutralization in the test stand by experiments and calculation.

Development of a strongly focusing high-intensity He(+) ion source for a confined alpha particle measurement at ITER.

A strongly focusing high-intensity He(+) ion source has been designed and constructed as a beam source for a high-energy He(0) beam probe system for diagnosis of fusion produced alpha particles in the thermonuclear fusion plasmas. The He(+) beam was extracted from the ion source at an acceleration voltage of 18-35 kV. Temperature distributions of the beam target were observed with an IR camera. The 1/e-holding beam profile half-width was about 15 mm at optimum perveance (Perv) of 0.03 (I(beam)=2.4 A). A beam current about 3 A was achieved at an acceleration voltage of 26.7 kV with an arc power of 10 kW (Perv=0.023).

A distinct function of the retinoblastoma protein in the control of lipid composition identified by lipidomic profiling.

Here, by combining lipidomics with transcriptome analysis, we demonstrate that Rb depletion in mouse embryonic fibroblastss induces significant alterations in their lipid composition. We discovered that Rb depletion induced increase in lysophosphatidylserine, diacylglycerol (DAG), fatty acid (FA), acylcarnitine, phosphatidylcholine (PC), arachidonoyl ethanolamine, and decrease in phosphatidylglycerol, monoacylglycerol, without change in total lipid per protein levels. Analysis of the acyl chain composition of DAG, PC and phosphatidylserine revealed increase of saturated and mono-unsaturated acyl chains with specific carbon chain length. Consistently, we observed that Rb depletion increased the levels of fatty acids with the corresponding carbon chain length and number of carbon-carbon double bondssuch as myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0) and all forms of FA 18:1. Microarray analysis revealed that Rb depletion induced significant upregulation of enzymes involved in elongation and desaturation of fatty acids. Among these, we found that elongation of long chain fatty acid family member 6 (Elovl6) and stearoyl-CoA desaturase 1 (Scd1) are the most robustly controlled by Rb possibly through E2F and sterol regulatory element-binding protein transcription factors. Depletion of Elovl6 or Scd1 significantly suppressed colony formation, sphere formation and xenograft tumor growth of Rb-deficient tumor cells. Suppression of self-renewal by the SCD1 inhibitor was rescued upon supplementation of the mono-unsaturated fatty acids generated by this enzyme. This study suggests a novel role for Rb in suppressing the malignant progression of tumors by controlling the lipid composition.

The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Biological behavior of cloned cells of human malignant fibrous histiocytoma in vivo and in vitro.

A human malignant fibrous histiocytoma cell line was established in vitro. Cells showed a wide variety of morphologies, although the karyotype study showed that the tumor was monoclonal in origin because of the presence of unique marker chromosomes in 100% of the cells examined (50 of 50). Cells were cloned according to their characteristic morphologies and biological behavior in culture. The cloned cells were sparse spindle, packed spindle, epithelioid, and lymphoid. In colonies, sparse spindle cells grew separately from each other without cell to cell contact but produced a cartwheel pattern at confluency. Packed spindle cells grew in a tightly packed fashion and produced a storiform pattern at confluency. Epithelioid cells were spindle shaped as individuals but became epithelioid when in contact with each other and produced many multinucleated giant cells at confluency. Lymphoid cells were spindle shaped as individuals but became spherical at confluency. When tumors were grown in nude mice after transplantation of these cloned cells, the histology was shown to be unrelated to morphology in culture and was epithelioid (histiocytic), as was the original tumor. These results show that (a) a single cell derived from malignant fibrous histiocytoma cells exhibits a wide range of phenotypical expression in vitro, (b) cells have their own morphological and biological characteristics in vitro, which (c) however, are easily influenced by environmental factors and (d) which are unstable and even interchangeable. These characteristics may contribute to the endless variety of cellular forms and growth patterns of malignant fibrous histiocytomas in humans.

High expression of S-phase kinase-interacting protein 2, human F-box protein, correlates with poor prognosis in oral squamous cell carcinomas.

Reduced expression of p27(Kip1), a cyclin-dependent kinase (Cdk) inhibitor, is frequently found in various cancers, including oral squamous cell carcinoma (OSCC), and is attributable to an enhancement of its degradation. Skp2, an F-box protein necessary for DNA replication, is required for the ubiquitinylation and subsequent degradation of p27(Kip1). In the present study, we examined the expression of Skp2 and its correlation with the expression of p27(Kip1) protein or p27(Kip1) degradation in OSCC. Using immunohistochemistry, we found that high expression of Skp2 was present in 49% of OSCCs and only 20% of epithelial dysplasias. Significantly, high expression of Skp2 was correlated with poor prognosis of OSCC patients. We also found an inverse correlation between the expression of Skp2 and p27 by immunohistochemical analysis. A similar correlation was observed in OSCC cell lines and OSCC tissues by Western blot analysis. Interestingly, OSCC tissues with Skp2 expression had high p27(Kip1) degradation activity. These findings indicate that (a) Skp2 may play an important role for the development of OSCC, (b) Skp2 can be a novel target for OSCC treatment as well as a strong prognostic marker, and (c) the reduction in p27(Kip1) protein may be brought about by enhancement of its degradation mediated by increased levels of Skp2 protein.

Involvement of a macromolecular activating factor in activity of guanylate cyclase partially purified from supernatant of a pig lung extract.

A guanylate cyclase preparation partially purified from supernatant of a pig lung extract was subjected to affinity chromatography on an Agarose-GTP column. The major portion of the cyclase activity was adsorbed on the column and then eluted with 50 mM EDTA and 0.5 M KCl, whereas the fractions non-adsorbed on the column contained a factor which enhanced the cyclase activity. Addition of the activating factor to a cyclase reaction mixture increase the enzyme activity without a time lag, and this enhancement by the factor was dose-dependent. With concomitant presence of cyclase and the factor in the reaction mixture the apparent Km value for GTP-Mn2+ of the enzyme was 56 microM, this value being the same as in absence of the factor, however, here the maximum velocity increased 4-fold. The factor was nondiffusable, heat-labile, partially sensitive to trypsin, and resistant to acid or alkali. As estimated by gel filtration, this factor had an apparent molecular weight of 85 000.