Oxidized LDL and lysophosphatidylcholine stimulate plasminogen activator inhibitor-1 expression through reactive oxygen species generation and ERK1/2 activation in 3T3-L1 adipocytes.

Plasminogen activator inhibitor-1 (PAI-1) is secreted from adipose tissue and is considered to be a risk factor for both atherosclerosis and insulin resistance. Here we report for the first time that PAI-1 expression is enhanced by oxidized low-density lipoprotein (OxLDL) and its lipid component lysophosphatidylcholine (LPC) in mouse 3T3-L1 adipocytes. In fully differentiated 3T3-L1 cells, OxLDL treatment increased the mRNA expression and protein secretion of PAI-1 in a dose- and time-dependent manner, whereas native LDL had no effect. The addition of an anti-CD36 antibody suppressed OxLDL-stimulated PAI-1 expression by 50%, suggesting that adipose-derived CD36 contributes to roughly half of the PAI-1 expression stimulated by OxLDL. In addition, pharmacological experiments showed that the OxLDL-stimulated enhancement in PAI-1 expression was mediated through the generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2. Furthermore, LPC, a major lipid component of OxLDL, was responsible for the enhanced expression of PAI-1 as phospholipase A(2)-treated acetyl LDL, which generates LPC, strongly stimulated PAI-1 expression, whereas acetyl LDL itself had no such activity. These data demonstrate that the uptake of OxLDL and, in particular, its lipid component LPC into adipocytes triggers aberrant ROS-mediated PAI-1 expression, which may be involved in the pathogenesis of metabolic syndrome.

Differential usage of alternate promoters of the human stress response gene ATF3 in stress response and cancer cells.

Stress response gene ATF3 plays a pleiotropic role in determining cell fate in response to mitogenic or stress stimuli. An alternate promoter of the human ATF3 gene (designated P1 in this study) has recently been reported, which is located approximately 43.5 kb upstream of the previously reported P2 promoter. We showed here that the P1 promoter is highly conserved between human and mouse and is functional in response to various stimuli, whereas the P1 promoter was dominantly induced by serum and the P2 promoter was more efficiently activated in response to TGF-beta and oncogenic HRAS. The P1 promoter contains multiple transcriptional start sites, and the different 5'-UTRs markedly affected their translation in response to stress. In human prostate and Hodgkin Reed-Sternberg cancer cells with elevated expression of ATF3, the P1 promoter was constitutively activated and its chromatin structure was modified into active configuration. The differential usage of alternate promoters of the ATF3 gene at both transcriptional and translational level and the modification of chromatin structure may provide a novel mechanism for expressing ATF3 in determining cell fate during stress response and cancer.

[Use of sugammadex in a patient with narrow angle glaucoma].

An 86-year-old woman was scheduled to receive fourth reconstructive surgery for femoral bone fracture under general anesthesia. She had been suspected with narrow angle glaucoma due to headache and bloodshot eyes during gastroscopy. During transfer to our hospital, she fell down and suffered from the right femoral neck fracture. The patient underwent femoral head replacement under spinal anesthesia. Later, she received surgeries twice uneventfully under spinal anesthesia; removal and re-implantation of the femoral bone head due to infection of the implanted head. Six months later, she fell down again and femoral bone was fractured during rehabilitation. Anesthesia was induced with propofol followed by rocuronium 0.9 mg x kg(-1) i.v. Anesthesia was maintained with propofol and remifentanil, and rocuronium was administered to maintain PTC of 10 or less. The surgery was completed in 150 minutes. At the end of surgery, a laryngeal mask was inserted and the tracheal tube was removed. TOF ratio recovered to 80% 8 minutes after sugammadex 2 mg kg(-1) i.v., and increased to 100% 3 minutes after additional 1 mg x kg(-1). Intraocular pressure stayed below 20 mmHg during the intervention. We could achieve full reversal of neuromuscular blockade and suppress increase in intraocular pressure with use of sugammadex.

[Propofol triggers a marked body temperature increase in a patient with fulminant malignant hyperthermia (MH) without inducing other symptoms of MH].

A 53-year-old woman who had experienced symptoms of fulminant malignant hyperthermia (MH) by sevoflurane a week before and her MH muscle biopsy revealing positive later, underwent the right hemicolectomy under total intravenous anesthesia with propofol and fentanyl. The patient's body temperature increased at a rate of 0.6 degree C per 15 min from 37.5 to 39.4 degrees C, but other symptoms of MH, such as tachycardia, arrhythmia, acidemia, and hypoxemia, were obviously slight in comparison with those induced by sevoflurane. The body temperature decreased after discontinuation of propofol and administration of dantrorene injection. When the patient received continuous propofol infusion for the purpose of sedation in the intensive care unit again, the body temperature gradually increased to 40 degrees C. However, it decreased to 37.8 degrees C after discontinuation of propofol and dantrorene injection again. It is well recognized that propofol is not a MH trigger, but it shoud be noted that some MH patients could experience a hypermetabolic state, such as hyperthermia, even by propofol.

[Spinal anesthesia in a patient with SMON disease].

We report a patient with subacute myelo-optico-neuropathy (SMON) in whom spinal anesthesia was employed to treat fracture of the femur neck. An 87-year-old woman was diagnosed as having SMON at the age of 45. The patient was admitted to our hospital with fracture of the femur neck. Aspiration pneumonia was also suspected with shadow in the right lung on the chest X-P The percutaneous oxygen saturation (Spo2) with room air was 77%. Spinal anesthesia with 5 mg of 0.5% hyperbaric bupivacaine and 20 mcg of fentanyl was performed at L3-4. The level of anesthesia was T4. During surgery, no severe pain in the lower limbs was observed. Three hours after the end of surgery, the level of anesthesia was T9. On the day after surgery, the extent of dysesthesia and reflex were similar to those before surgery. General anesthesia has been chosen in SMON patients, because there was a report of severe pain of the lower limbs after spinal anesthesia with dibucaine. In our patient, general anesthesia was considered inappropriate due to hypoxemia. We used a mixture of bupivacaine and fentanyl for spinal anesthesia, because the neurotoxicity of bupivacaine is weaker than that of dibucaine.

[Combined use of the airway scope and fiberoptic bronchoscopy for tracheal intubation in a patient with a large epiglottic cyst].

A 26-year-old man was scheduled for surgical extraction of a large epiglottic cyst. Mask ventilation was possible under propofol anesthesia without muscle relaxant. It was difficult to see the glottis using either a Macintosh laryngoscope or by fiberoptic bronchoscopy. When the AWS laryngoscope (Hoya, Tokyo Japan) with a part of the blade removed, was inserted orally, it became possible to see the glottis with a part of the epiglottic cyst. A reinforced tube was inserted nasally, and a fiberoptic bronchoscope was passed through the tube into the trachea. The tube was then passed over the fiberscope into the trachea. We believe that the Pentax AWS laryngoscope may lift the epiglottis and its cyst atraumatically, and may facilitate nasal fiberoptic intubation in a patient with a large epiglottic cyst.

Mould filling of Ag-Pd-Cu-Au and Ag-Zn-Sn-In alloy castings made using a rapidly prepared gypsum-bonded investment material.

Mandibular premolar-shaped wax patterns of full crowns with a marginal angle of 300 were prepared. Two semiprecious alloys were cast using a rapidly prepared gypsum-bonded investment material or a conventional gypsum-bonded investment. A precise impression was taken and cut into four segments. Scanning electron microscopy was used to evaluate the mould filling of each segment. The mould filling of the silver-palladium-copper-gold alloy was worse than that of the silver-zinc-tin-indium alloy. The mould filling of both alloys cast with the rapidly prepared gypsum-bonded investment material was superior to that using the conventional investment.

Acetylcholine released from T cells regulates intracellular Ca2+, IL-2 secretion and T cell proliferation through nicotinic acetylcholine receptor.

AIMS: T lymphocytes synthesize acetylcholine (ACh) and express muscarinic and nicotinic ACh receptors (mAChR and nAChR, respectively) responsible for increases in the intracellular Ca2+ concentration ([Ca2+]i). Our aim in the present study was to assess whether autocrine ACh released from T lymphocytes regulates their physiological functions. MAIN METHODS: MOLT-3 human leukemic cell line and murine splenocytes were loaded with fura-2 to monitor [Ca2+]i changes in the absence or presence of several AChR antagonists, including mecamylamine, methyllycaconitine and scopolamine. Real-time PCR and ELISA were performed to measure interleukin-2 (IL-2) mRNA and protein levels. KEY FINDINGS: T lymphocytes constitutively produce sufficient amounts of ACh to elicit autocrine changes in [Ca2+]i. These autocrine ACh-evoked [Ca2+]i transients were mediated by nAChRs and then influx of extracellular Ca2+. Mecamylamine, a nAChR inhibitor, suppressed not only these [Ca2+]i transients, but also IL-2 release and T cell proliferation. SIGNIFICANCE: Here, we confirmed that T lymphocytes utilize ACh as a tool to interact with each other and that autocrine ACh-activated nAChRs are involved in cytokine release and cell proliferation. These findings suggest the possibility that nAChR agonists and antagonists and smoking are able to modulate immune function, which in turn suggests the therapeutic potential of immune activation or suppression using nAChR agonists or antagonists.

Effects of amino acids on the formation of hematite particles in a forced hydrolysis reaction.

The influence of amino acids on the formation of hematite particles from a forced hydrolysis reaction of acidic FeCl3 solution was examined. The spherical particles were produced on the systems with L-phenylalanine (L-Phe), L-serine (L-Ser) and L-alanine (L-Ala), though L-glutamine (L-Gln) and L-glutamic acid (L-Glu) gave ellipsoidal hematite particles. This morphological change in hematite particles is consistent with the order of stability constant of amino acids against to Fe3+ ions (K). The hematite particles produced with L-Glu, L-Gln and L-Ser were highly porous because they are formed by aggregation of cluster particles. These particles exhibited microporous behavior by outgassing the particles below 200 degrees C while they changed to mesoporous after treating above 300 degrees C by elimination of amino acids molecules remained between the cluster particles within the hematite particles. The hematite particles strongly depended on the nature of amino acids such as alternation of solution pH and adsorption affinity to beta-FeOOH and/or polynuclear primary (PN) particles. The systems on L-Ala and L-Phe, showing very rapid phase transformation from beta-FeOOH to hematite, exhibited the Ostwald ripening. A rotational particle preparation procedure suggested that the morphology of hematite particle is governed by the mode and strength of amino acid adsorption onto beta-FeOOH and/or PN particles.

Systems analysis of ATF3 in stress response and cancer reveals opposing effects on pro-apoptotic genes in p53 pathway.

Stress-inducible transcription factors play a pivotal role in cellular adaptation to environment to maintain homeostasis and integrity of the genome. Activating transcription factor 3 (ATF3) is induced by a variety of stress and inflammatory conditions and is over-expressed in many kinds of cancer cells. However, molecular mechanisms underlying pleiotropic functions of ATF3 have remained elusive. Here we employed systems analysis to identify genome-wide targets of ATF3 that is either induced by an alkylating agent methyl methanesulfonate (MMS) or over-expressed in a prostate tumour cell line LNCaP. We show that stress-induced and cancer-associated ATF3 is recruited to 5,984 and 1,423 targets, respectively, in the human genome, 89% of which are common. Notably, ATF3 targets are highly enriched for not only ATF/CRE motifs but also binding sites of several other stress-inducible transcription factors indicating an extensive network of stress response factors in transcriptional regulation of target genes. Further analysis of effects of ATF3 knockdown on these targets revealed that stress-induced ATF3 regulates genes in metabolic pathways, cell cycle, apoptosis, cell adhesion, and signalling including insulin, p53, Wnt, and VEGF pathways. Cancer-associated ATF3 is involved in regulation of distinct sets of genes in processes such as calcium signalling, Wnt, p53 and diabetes pathways. Notably, stress-induced ATF3 binds to 40% of p53 targets and activates pro-apoptotic genes such as TNFRSF10B/DR5 and BBC3/PUMA. Cancer-associated ATF3, by contrast, represses these pro-apoptotic genes in addition to CDKN1A/p21. Taken together, our data reveal an extensive network of stress-inducible transcription factors and demonstrate that ATF3 has opposing, cell context-dependent effects on p53 target genes in DNA damage response and cancer development.

Suppression of fibroblast growth factor-2 expression: possible mechanism underlying methylmercury-induced inhibition of the repair of wounded monolayers of cultured human brain microvascular endothelial cells.

Vascular toxicity is an important feature of the neuropathy induced by methylmercury. Methylmercury does not cause nonspecific cell damage, but rather retards the repair of wounded monolayers of cultured human brain microvascular endothelial cells by inhibiting their proliferation. Since vascular endothelial cell proliferation during the repair process strongly depends on the fibroblast growth factor-2 (FGF-2) system, we investigated the effects of methylmercury on the expression of FGF-2 and related proteins (i.e., FGF receptor 1 and perlecan) in cultured human brain microvascular endothelial cells. Of the mRNAs examined, FGF-2 mRNA expression was significantly lowered by methylmercury in not only wounded monolayers but also dense and sparse cultures of endothelial cells; a lower expression of FGF-2 protein in the cells was confirmed. In addition, exogenous FGF-2 partially abrogated the proliferation-inhibitory effect of methylmercury. The results of this study suggest that suppression of FGF-2 expression is one of the mechanisms underlying the inhibitory effect of methylmercury in damaged endothelial cell monolayers. The FGF-2 system may be one of the important biological systems behind the vascular toxicity of methylmercury.