The Quinazoline Otaplimastat (SP-8203) Reduces the Hemorrhagic Transformation and Mortality Aggravated after Delayed rtPA-Induced Thrombolysis in Cerebral Ischemia.

Acute ischemic stroke is the leading cause of morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only agent clinically approved by FDA for patients with acute ischemic stroke. However, delayed treatment of rtPA (e.g., more than 3 h after stroke onset) exacerbates ischemic brain damage by causing intracerebral hemorrhage and increasing neurotoxicity. In the present study, we investigated whether the neuroprotant otaplimastat reduced delayed rtPA treatment-evoked neurotoxicity in male Sprague Dawley rats subjected to embolic middle cerebral artery occlusion (eMCAO). Otaplimastat reduced cerebral infarct size and edema and improved neurobehavioral deficits. In particular, otaplimastat markedly reduced intracerebral hemorrhagic transformation and mortality triggered by delayed rtPA treatment, consequently extending the therapeutic time window of rtPA. We further found that ischemia-evoked extracellular matrix metalloproteases (MMPs) expression was closely correlated with cerebral hemorrhagic transformation and brain damage. In ischemic conditions, delayed rtPA treatment further increased brain injury via synergistic expression of MMPs in vascular endothelial cells. In oxygen-glucose-deprived endothelial cells, otaplimastat suppressed the activity rather than protein expression of MMPs by restoring the level of tissue inhibitor of metalloproteinase (TIMP) suppressed in ischemia, and consequently reduced vascular permeation. This paper shows that otaplimastat under clinical trials is a new drug which can inhibit stroke on its own and extend the therapeutic time window of rtPA, especially when administered in combination with rtPA.

Sudachinoid- and Ichangensin-Type Limonoids from Citrus junos Downregulate Pro-Inflammatory Cytokines.

Limonoids, a dominant group of phytochemicals in the Rutaceae family, are known to exhibit several pharmacological activities. To identify natural products having efficacy against inflammatory bowel disease (IBD), we isolated 13 limonoids including a new compound, methyl sudachinoid A, from the seeds of Citrus junos and investigated their anti-inflammatory effects by assessing the expression of pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages and HT-29 human colon epithelial cells. Our findings revealed that limonoids significantly downregulated the pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α, and nuclear transcription factor κB. In particular, sudachinoid-type compounds, methyl sudachinoid A and sudachinoid B, and ichangensin-type compound, 1-O-methyichangensin downregulated the expression of pro-inflammatory cytokines more potently than other limonoids, nomilin and limonin, which have been previously reported to exhibit anti-inflammatory activities in other cells; nomilin and limonin were therefore employed as positive controls in this study. Herein, we reveal that the anti-inflammatory activities of limonoids including a new compound methyl sudachinoid A from C. junos were mediated via the downregulation of pro-inflammatory cytokines and these limonoids can be employed as potential therapeutic phytochemicals for IBD.

A Novel CD147 Inhibitor, SP-8356, Attenuates Pathological Fibrosis in Alkali-Burned Rat Cornea.

The corneal fibrotic responses to corneal damage often lead to severe corneal opacification thereby resulting in severe visual impairment or even blindness. The persistence of corneal opacity depends heavily on the activity of corneal myofibroblast. Myofibroblasts are opaque and synthesize a disorganized extracellular matrix (ECM) and thus promoting opacification. Cluster of differentiation 147 (CD147), a member of the immunoglobulin superfamily, is known to play important roles in the differentiation process from fibroblast to myofibroblast in damaged cornea and may therefore be an effective target for treatment of corneal opacity. Here, we examined the therapeutic efficacy of novel CD147 inhibiting verbenone derivative SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) on corneal fibrosis. Topical SP-8356 significantly reduced corneal haze and fibrosis in the alkali-burned cornea. In detail, SP-8356 inhibited both alpha-smooth muscle actin (α-SMA) expressing myofibroblast and its ECM-related products, such as matrix-metalloproteinase-9 and collagen type III and IV. Similar to SP-8356, topical corticosteroid (prednisolone acetate, PA) also reduced the ECM-related products and opacification. However, prednisolone acetate failed to decrease the population of α-SMA-positive corneal myofibroblast. In conclusion, SP-8356 is capable enough to prevent corneal haze by preventing pathological fibrosis after severe corneal damage. Therefore, SP-8356 could be a potentially promising therapeutic drug for corneal fibrosis.

A novel CD147 inhibitor, SP-8356, reduces neointimal hyperplasia and arterial stiffness in a rat model of partial carotid artery ligation.

BACKGROUND: Neointimal hyperplasia and its related arterial stiffness are the crucial pathophysiological features in atherosclerosis and in-stent restenosis. Cluster of differentiation 147 (CD147), a member of the immunoglobulin super family that induces the expression of matrix metalloproteinase-9 (MMP-9) by dimerization, may play important roles in neointimal hyperplasia and may therefore be an effective target for the treatment of this condition. Here, we investigated whether a novel CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) reduces neointimal hyperplasia and arterial stiffness in a rat model of partial carotid artery ligation. METHODS: Neointimal hyperplasia was induced in Sprague-Dawley rats by partial ligation of the right carotid artery combined with a high fat diet and vitamin D injection. Rats were subdivided into vehicle, SP-8356 (50 mg/kg), and rosuvastatin (10 mg/kg) groups. The drugs were administrated via intraperitoneal injections for 4 weeks. The elasticity of blood vessels was assessed by measuring pulse wave velocity using Doppler ultrasonography before sacrifice. Histomolecular analysis was carried out on harvested carotid arteries. RESULTS: SP-8356 significantly reduced MMP activity by inhibiting CD147 dimerization. SP-8356 reduced neointimal hyperplasia and prevented the deterioration of vascular elasticity. SP-8356 had a greater inhibitory effect on neointimal hyperplasia than did rosuvastatin. Furthermore, rosuvastatin did not improve vascular elasticity. SP-8356 increased the expression of smooth muscle myosin heavy chain (SM-MHC), but decreased the expression of collagen type III and MMP-9 in the neointimal region. In contrast to SP-8356, rosuvastatin did not alter the expression of SM-MHC or MMP-9. CONCLUSIONS: The ability of SP-8356 to reduce neointimal hyperplasia and improve arterial stiffness in affected carotid artery suggests that SP-8356 could be a promising therapeutic drug for vascular remodeling disorders involving neointimal hyperplasia and arterial stiffness.

SP-8356, a Novel Inhibitor of CD147-Cyclophilin A Interactions, Reduces Plaque Progression and Stabilizes Vulnerable Plaques in apoE-Deficient Mice.

Interactions between CD147 and cyclophilin A (CypA) promote plaque rupture that causes atherosclerosis-related cardiovascular events, such as myocardial infarction and stroke. Here, we investigated whether SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one), a novel drug, can exert therapeutic effects against plaque progression and instability through disruption of CD147-CypA interactions in apolipoprotein E-deficient (ApoE KO) mice. Immunocytochemistry and immunoprecipitation analyses were performed to assess the effects of SP-8356 on CD147-CypA interactions. Advanced plaques were induced in ApoE KO mice via partial ligation of the right carotid artery coupled with an atherogenic diet, and SP-8356 (50 mg/kg) orally administrated daily one day after carotid artery ligation for three weeks. The anti-atherosclerotic effect of SP-8356 was assessed using histological and molecular approaches. SP-8356 interfered with CD147-CypA interactions and attenuated matrix metalloproteinase-9 activation. Moreover, SP-8356 induced a decreased in atherosclerotic plaque size in ApoE KO mice and stabilized plaque vulnerability by reducing the necrotic lipid core, suppressing macrophage infiltration, and enhancing fibrous cap thickness through increasing the content of vascular smooth muscle cells. SP-8356 exerts remarkable anti-atherosclerotic effects by suppressing plaque development and improving plaque stability through inhibiting CD147-CypA interactions. Our novel findings support the potential utility of SP-8356 as a therapeutic agent for atherosclerotic plaque.

Anti-Inflammatory Activities of Isogosferol, a Furanocoumarin Isolated from Citrus junos Seed Shells through Bioactivity-Guided Fractionation.

Citrus junos Tanaka is a traditional medicine for treating coughs, dyspepsia, diabetes, asthma, neuralgia, and inflammatory disorders, and is distributed in Asia, especially in Korea, Japan, and China. This study aimed to use bioactivity-guided fractionation to find therapeutic phytochemicals from C. junos seeds, which can attenuate inflammatory responses. Nine coumarins (1-9) were isolated from the methanolic extract of C. junos seed shells and the inhibitory effects against inflammatory mediators were investigated using murine macrophages. Among the coumarins, compound 3, isogosferol (ISO), more potently attenuated the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. ISO also inhibited the expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated macrophages. Additionally, the phosphorylation of extracellular-regulated kinases (pERK)1/2 was reduced by ISO. We confirmed that ISO attenuated the release of interleukin-1 beta (IL-1ß), which is a central mediator of the inflammatory response. These results demonstrate that ISO from C. junos seed shells may be a potent therapeutic candidate for the treatment of inflammatory diseases.

DISTINCTIVE LOCALIZATION OF GROUP 3 LATE EMBRYOGENESIS ABUNDANT SYNTHESIZING CELLS DURING BRINE SHRIMP DEVELOPMENT.

Despite numerous studies on late embryogenesis abundant (LEA) proteins, their functions, roles, and localizations during developmental stages in arthropods remain unknown. LEA proteins protect crucial proteins against osmotic stress during the development and growth of various organisms. Thus, in this study, fluorescence in situ hybridization was used to determine the crucial regions protected against osmotic stress as well as the distinctive localization of group 3 (G3) LEA(+) cells during brine shrimp development. Several cell types were found to synthesize G3 LEA RNA, including neurons, muscular cells, APH-1(+) cells, and renal cells. The G3 LEA(+) neuronal cell bodies outside of the mushroom body projected their axonal bundles to the central body, but those inside the mushroom body projected their axonal bundles toward the deutocerebrum without innervating the central body. The cell bodies inside the mushroom body received axons of the G3 LEA(+) sensory cells at the medial ventral cup of the nauplius eye. Several glands were found to synthesize G3 LEA RNA during the nauplius stages of brine shrimp, including the sinus, antennal I and II, salt, and three ectodermal glands. This study provides the first demonstration of the formation of G3 LEA(+) sinus glands at the emergence stages of brine shrimp. These results suggest that G3 LEA protein is synthesized in several cell types. In particular, specific glands play crucial roles during the emergence and nauplius stages of brine shrimp.

Gel-based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell.

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Silkworm 30K protein inhibits ecdysone-induced apoptosis by blocking the binding of ultraspiracle to ecdysone receptor-B1 in cultured Bm5 cells.

This study investigates the mechanism through which increased 30K protein inhibits ecdysone-induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20-hydroxyecdysone (20E) after transfection with the pIZT/V5-His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5-His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti-30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5-His-transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor-B1 (EcR-B1). Anti-30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5-His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR-B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR-B1-containing complexes from Bm5 cells transfected with control pIZT/V5-His vector and treated with 20E showed that EcR-B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5-His-transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR-B1 through formation of a 30K/EcR-B1 complex, resulting in inhibition of 20E-induced Bm5 cell apoptosis.

Xanthogranulomatous pancreatitis presents as a solid tumor mass: a case report.

Xanthogranulomatous inflammation (XGI) is a rare, idiopathic process in which lipid-laden histiocytes are deposited at various locations in the body. Although XGI has been reported to occur in various organs such as the gallbladder, kidney, bone, stomach, colon, appendix, lymph nodes, urachus, and urinary bladder and in soft tissues, xanthogranulomatous pancreatitis (XGP) is extremely rare. Herein, we report a case of XGP occurring in a 70-yr-old woman, who presented with abdominal pain for several months. On physical examination, mild epigastric tenderness was noted. Abdomen CT scan revealed a low attenuated mass in uncinate process of pancreas, suggesting malignant lesion. Whipple's operation was performed and the final pathologic diagnosis was XGP. The patient's post-operative course was uneventful, and no recurrence was found within 7 months of the operation. When a pancreatic mass does not show clinico-radiological features typical of common pancreatic neoplasms, XGP should be considered for a differential diagnosis.

Bioactivity-Guided Extract Optimization of Osmanthus fragrans var. aurantiacus Leaves and Anti-Inflammatory Activities of Phillyrin.

The aim of this study was to identify the optimal extraction conditions for leaves of Osmanthus fragrans var. aurantiacus. Inhibitory effects of various extracts on NO production were compared. Antioxidant evaluations for total phenol and flavonoid contents were carried out using various extracts of O. fragrans var. aurantiacus leaves obtained under optimal extraction conditions that showed the greatest effect on NO production. The optimal method for extracting O. fragrans var. aurantiacus leaves resulted in an extract named OP OFLE. OP OFLE showed DPPH and ABTS radical scavenging activities in a concentration-dependent manner. Phillyrin (PH) was isolated as a major compound from OP OFLE by HPLC/DAD analysis. OP OFLE and PH reduced inducible nitric oxide (iNOS) and cyclooxygenase (COX)-2 protein expression and downregulated proinflammatory cytokines such as interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 and HT-29 cells. To determine the signal pathway involved in the inhibition of NO production, a Western blot analysis was performed. Results showed that OP OFLE decreased phosphorylation of extracellular regulated kinase (pERK) 1/2 and the expression of nuclear factor-kappa B (NF-κB). Our results suggest that extracts of O. fragrans var. aurantiacus leaves and its major components have biological activities such as antioxidative and anti-inflammatory properties.

p-Hydroxybenzoic Acid ß-d-Glucosyl Ester and Cimidahurinine with Antimelanogenesis and Antioxidant Effects from Pyracantha angustifolia via Bioactivity-Guided Fractionation.

This study evaluated bioactivity-guided fractionation as a means to identify therapeutic phytochemicals from Pyracantha angustifolia that can attenuate melanogenesis and oxidation. Seven compounds with inhibitory effects on melanin production and tyrosinase (TYR) activity, and ABTS and DPPH radical-scavenging activities, which have not been reported as whitening materials, were isolated from the n-butanol fraction from P. angustifolia leaves (PAL). Among the seven compounds, p-hydroxybenzoic acid ß-d-glucosylester (HG), and cimidahurinine (CH) had strong inhibitory effects on melanin production and TYR activity, as well as ABTS and DPPH radical-scavenging activities. Western blot analysis showed that HG and CH suppressed tyrosinase-related protein (TYRP)-1 and TYRP-2 expression. Moreover, HG and CH inhibited reactive oxygen species (ROS) generation in tert-butyl hydroperoxide (t-BHP)-treated B16F10 cells. These results suggest that P. angustifolia containing active compounds, such as HG and CH, is a potent therapeutic candidate for the development of hypopigmenting agents.

Calpain-mediated cleavage of Fbxw7 during excitotoxicity.

Neuronal cell death induced by ischemic injury has been attributed to glutamate receptor-mediated excitotoxicity, which is known to be accompanied by Ca2+ overload in the cytoplasm with concomitant activation of calcium-dependent mechanisms. More specifically, the overactivation of calpains, calcium-dependent cysteine proteases, have been associated with neuronal cell death following glutamate treatment. Previously, we observed decreased expression levels of F-box/WD repeat domain-containing protein 7 (Fbxw7) after the hyperactivation of cyclin-dependent kinase 5 (Cdk5) in cortical neurons challenged with glutamate. As determined using in vitro calpain cleavage assays, we demonstrated that the cleavage of Fbxw7 was mediated by activated calpain and attenuated in the presence of the calpain inhibitor, calpeptin. Using the rat middle cerebral artery occlusion model, we confirmed that Fbxw7 was indeed cleaved by activated calpain in the ipsilateral cortex. Based on our data, we hypothesize that the negative regulation of Fbxw7 by calpain may contribute to neuronal cell death and that the preservation of Fbxw7 by the inhibition of calpain, Cdk5, or both composes a novel protective mechanism following excitotoxicity.

Post-ischemic treatment of WIB801C, standardized Cordyceps extract, reduces cerebral ischemic injury via inhibition of inflammatory cell migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Anti-inflammatory therapy has been intensively investigated as a potential strategy for treatment of cerebral stroke. However, despite many positive outcomes reported in animal studies, anti-inflammatory treatments have not proven successful in humans as yet. Although immunomodulatory activity and safety of Cordyceps species (Chinese caterpillar fungi) have been proven in clinical trials and traditional Asian prescriptions for inflammatory diseases, its anti-ischemic effect remains elusive. AIM OF THE STUDY: In the present study, therefore, we investigated the potential therapeutic efficacy of WIB801C, the standardized extract of Cordyceps militaris, for treatment of cerebral ischemic stroke. MATERIALS AND METHODS: The anti-chemotactic activity of WIB801C was assayed in cultured rat microglia/macrophages. Sprague-Dawley rats were subjected to ischemic stroke via either transient (1.5-h tMCAO and subsequent 24-h reperfusion) or permanent middle cerebral artery occlusion (pMCAO for 24-h without reperfusion). WIB801C was orally administered twice at 3- and 8-h (50mg/kg each) after the onset of MCAO. Infarct volume, edema, blood brain barrier and white matter damages, neurological deficits, and long-term survival rates were investigated. The infiltration of inflammatory cells into ischemic lesions was assayed by immunostaining. RESULTS: WIB801C significantly decreased migration of cultured microglia/macrophages. This anti-chemotactic activity of WIB-801C was not mediated via adenosine A3 receptors, although cordycepin, the major ingredient of WIB801C, is known as an adenosine receptor agonist. Post-ischemic treatment with WIB801C significantly reduced the infiltration of ED-1-and MPO-positive inflammatory cells into ischemic lesions in tMCAO rats. WIB801C-treated rats exhibited significantly decreased infarct volume and cerebral edema, less white matter and blood-brain barrier damages, and improved neurological deficits. WIB801C also improved survival rates over 34 days after ischemia onset. A significant reduction in infarct volume and neurobehavioral deficits by WIB801C was also observed in rats subjected to pMCAO. CONCLUSIONS: In summary, post-ischemic treatment of WIB801C reduced infiltration of inflammatory cells into ischemic lesions via inhibition of chemotaxis, which confers long-lasting histological and neurological protection in ischemic brain. WIB801C may be a promising anti-ischemic drug candidate with clinically relevant therapeutic time window and safety.

Simvastatin Reduces Lipopolysaccharides-Accelerated Cerebral Ischemic Injury via Inhibition of Nuclear Factor-kappa B Activity.

Preceding infection or inflammation such as bacterial meningitis has been associated with poor outcomes after stroke. Previously, we reported that intracorpus callosum microinjection of lipopolysaccharides (LPS) strongly accelerated the ischemia/reperfusion-evoked brain tissue damage via recruiting inflammatory cells into the ischemic lesion. Simvastatin, 3-hydroxy-3-methylgultaryl (HMG)-CoA reductase inhibitor, has been shown to reduce inflammatory responses in vascular diseases. Thus, we investigated whether simvastatin could reduce the LPS-accelerated ischemic injury. Simvastatin (20 mg/kg) was orally administered to rats prior to cerebral ischemic insults (4 times at 72, 48, 25, and 1-h pre-ischemia). LPS was microinjected into rat corpus callosum 1 day before the ischemic injury. Treatment of simvastatin reduced the LPS-accelerated infarct size by 73%, and decreased the ischemia/reperfusion-induced expressions of pro-inflammatory mediators such as iNOS, COX-2 and IL-1ß in LPS-injected rat brains. However, simvastatin did not reduce the infiltration of microglial/macrophageal cells into the LPS-pretreated brain lesion. In vitro migration assay also showed that simvastatin did not inhibit the monocyte chemoattractant protein-1-evoked migration of microglial/macrophageal cells. Instead, simvastatin inhibited the nuclear translocation of NF-κB, a key signaling event in expressions of various proinflammatory mediators, by decreasing the degradation of IκB. The present results indicate that simvastatin may be beneficial particularly to the accelerated cerebral ischemic injury under inflammatory or infectious conditions.

4-hydroxy-2(E)-Nonenal facilitates NMDA-Induced Neurotoxicity via Triggering Mitochondrial Permeability Transition Pore Opening and Mitochondrial Calcium Overload.

N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is one of the major causes for neuronal cell death during cerebral ischemic insult. Previously, we reported that the final product of lipid membrane peroxidation 4-hydroxy-2E-nonenal (HNE) synergistically increased NMDA receptor-mediated excitotoxicity (J Neurochem., 2006). In this study, we investigated the mechanism involved in the synergistic neuronal cell death induced by co-treatment with HNE and NMDA. Although neither HNE (1 µM) nor NMDA (2 µM) alone induced the death of cortical neurons, simultaneous treatment of neuronal cells with HNE and NMDA synergistically evoked the death of the cells. However, the synergistic effect on neuronal death was observed only in the presence of calcium. HNE neither increased the cytosolic calcium level ([Ca(2+)]i) nor altered the NMDA-induced intracellular calcium influx. However, HNE together with NMDA elevated the mitochondrial calcium level and depolarized the mitochondrial transmembrane potential. Furthermore, HNE evoked damage of isolated mitochondria at the cytosolic calcium level (200 nM), which is maximally induced by 2 µM NMDA. Consistently, ATP was depleted in neurons when treated with both HNE and NMDA together. Ciclopirox, a potent inhibitor of mitochondrial permeability transition pore opening (Br. J. Pharmacol., 2005), largely prevented the synergistic damage of mitochondria and death of cortical neurons. Therefore, although low concentrations of HNE and NMDA cannot individually induce neuronal cell death, they can evoke the neuronal cell death by synergistically accelerating mitochondrial dysfunction.

Risk factors of cryptogenic hepatocellular carcinoma in patients with low body mass index or without metabolic syndrome.

BACKGROUND/AIMS: Many patients are diagnosed with cryptogenic hepatocellular carcinoma (HCC) without metabolic syndrome (MS). We investigated the risk factors for cryptogenic HCC in patients with a low body mass index (BMI) or without MS. METHODS: Thirty-six patients were diagnosed with cryptogenic HCC over a 10-year period at a tertiary research hospital. Data including BMI score and risk factors for MS were analyzed retrospectively. Patients with fewer than two risk factors for MS (n = 16) were compared with those with two or more risk factors (n = 20). Patients with high BMI (≥ 23 kg/m(2), n = 20) were also compared with those with lower BMI (n = 16). RESULTS: Patients with fewer than two risk factors for MS were significantly more likely to smoke and be hepatitis B surface antibodies (anti-HBs)-positive vs. patients with two or more risk factors. However, only smoking was statistically significant on multivariate analysis. Peaks of BMI were observed in two regions. Lower BMI was significantly associated with the presence of anti-HBs compared with high BMI, although this association was not statistically significant on multivariate analysis. CONCLUSIONS: Smoking is a potential risk factor for cryptogenic HCC in patients without MS. Remote hepatitis B virus infection may be a risk factor for cryptogenic HCC in patients without MS or with a low BMI.

Increase of 30K protein in identified motoneurons by hemolymph results in inhibition of programmed cell death in silkworm, Bombyx mori (Lepidoptera, Bombycidae).

This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph.

Comparative evaluation of hypoxic-ischemic brain injury by flow cytometric analysis of mitochondrial membrane potential with JC-1 in neonatal rats.

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic-ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V(+)/PI(+) necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V(-)/PI(-) live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

Risk Factors Associated with Left Ventricular Diastolic Dysfunction in Type 2 Diabetic Patients without Hypertension.

BACKGROUND: Hypertension and age are recognized as important risk factors for left ventricular (LV) diastolic dysfunction. Some studies have shown that diabetes itself may also be an independent risk factor for LV diastolic dysfunction, although this is controversial. The aim of this study was to determine the factors associated with LV diastolic dysfunction in patients with type 2 diabetes in the absence of hypertension or ischemic heart disease (IHD). METHODS: Participants in this study consisted of 65 type 2 diabetes patients (M : F = 45 : 20; mean age 51 [26 to 76] years; mean body mass index [BMI] 25.0 +/- 2.5 kg/m(2)) without hypertension, heart disease, or renal disease. Individuals with ischemic electrocardiographic changes were excluded. LV diastolic function was evaluated by Doppler echocardiographic studies. RESULTS: Fifteen patients (23.1%) showed LV diastolic dysfunction on Doppler echocardiographic studies. Patients with LV diastolic dysfunction were older than those without diastolic dysfunction (60.0 +/- 2.5 vs. 50.5 +/- 1.9 years; P < 0.01). After adjusting for age and sex, BMI was higher (26.6 +/- 0.7 vs. 24.6 +/- 0.3 kg/m(2); P < 0.01) and diabetes duration was longer (9.65 +/- 1.48 vs. 4.71 +/- 0.78 years; P < 0.01) in patients with LV diastolic dysfunction than in those without diastolic dysfunction. There were no differences in sex, smoking, blood pressure, lipid profiles, hemoglobin A(1)C, fasting glucose, fasting insulin, or diabetic microvascular complications between the LV diastolic dysfunction group and the normal diastolic function group. After adjusting for age, sex, and BMI, diabetes duration was found to be independently associated with LV diastolic dysfunction (odds ratio 1.38; confidence interval 1.12 to 1.72; P = 0.003). CONCLUSION: These results suggest that diabetes duration may be a risk factor for LV diastolic dysfunction in type 2 diabetic patients without hypertension or IHD.