Spatial summation of thermal sensitivity is limited to small areas: Comparisons of the forehead, forearm, abdomen, and foot.

The purpose of the present study was to examine if spatial summation in thermal sensitivity exists when stimulating areas larger than about 1% of body surface area (BSA) (approximately 200 cm2). We hypothesized that spatial summation would exist within a limited area and the effect would be insignificant for over the 1%BSA. Fifteen young males participated in this study and we measured their warmth and hot sensation thresholds on the four body regions (the forehead, forearm, abdomen, and instep) using the three sizes of radiant film heaters (10 × 10, 15 × 15, and 20 × 20 cm2 heating film area). The heating panel was kept at a distance of 10 cm from the skin and the surface temperature of the heating panel increased by 1 °C·s-1. The results showed that warmth and hot sensation thresholds were higher for the 100 cm2 condition than the 225 or 400 cm2 conditions (P < 0.05), but no differences were found between the 225 and 400 cm2 conditions. Secondly, the instep was most insensitive to the gradual increase of radiant heat among the four body regions for all three stimulating film sizes, even though the hot threshold was lowest for the instep because the initial foot temperature was lower than other skin temperatures. In summary, spatial summation in thermal sensitivity was found for the 100 and 225 cm 2 conditions, but not for the 225 and 400 cm2 conditions. These results suggest that spatial summation exists but limited to small stimulating areas, smaller than approximately 1% BSA.

Inhibition of DNMT3B and PI3K/AKT/mTOR and ERK Pathways as a Novel Mechanism of Volasertib on Hypomethylating Agent-Resistant Cells.

Resistance to hypomethylating agents (HMAs) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is a concerning problem. Polo-like kinase 1 (PLK1) is a key cell cycle modulator and is known to be associated with an activation of the PI3K pathway, which is related to the stabilization of DNA methyltransferase 1 (DNMT1), a target of HMAs. We investigated the effects of volasertib on HMA-resistant cell lines (MOLM/AZA-1 and MOLM/DEC-5) derived from MOLM-13, and bone marrow (BM) samples obtained from patients with MDS (BM blasts >5%) or AML evolved from MDS (MDS/AML). Volasertib effectively inhibited the proliferation of HMA-resistant cells with suppression of DNMTs and PI3K/AKT/mTOR and ERK pathways. Volasertib also showed significant inhibitory effects against primary BM cells from patients with MDS or MDS/AML, and the effects of volasertib inversely correlated with DNMT3B expression. The DNMT3B-overexpressed AML cells showed primary resistance to volasertib treatment. Our data suggest that volasertib has a potential role in overcoming HMA resistance in patients with MDS and MDS/AML by suppressing the expression of DNMT3 enzymes and PI3K/AKT/mTOR and ERK pathways. We also found that DNMT3B overexpression might be associated with resistance to volasertib.

Antileukemic activity of YPN-005, a CDK7 inhibitor, inducing apoptosis through c-MYC and FLT3 suppression in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive blood cancer with a high rate of relapse associated with adverse survival outcomes, especially in elderly patients. An aberrant expression of cyclin dependent kinase 7 (CDK7) is associated with poor outcomes and CDK7 inhibition has showed antitumor activities in various cancers. We investigated the efficacy of YPN-005, a CDK7 inhibitor in AML cell lines, xenograft mouse model, and primary AML cells. YPN-005 effectively inhibited the proliferation of AML cells by inducing apoptosis and reducing phosphorylation of RNA polymerase II. The c-MYC expression decreased with treatment of YPN-005, and the effect of YPN-005 was negatively correlated with c-MYC expression. YPN-005 also showed antileukemic activities in primary AML cells, especially those harboring FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation and in in vivo mouse model. Phosphorylated FLT3/Signal transducer and activator of transcription 5 (STAT5) was decreased and FLT3/STAT5 was downregulated with YPN-005 treatment. Our data suggest that YPN-005 has a role in treating AML by suppressing c-MYC and FLT3.

Expression and prognostic significance of microRNAs in Korean patients with myelodysplastic syndrome.

BACKGROUND/AIMS: Various alterations of microRNA (miRNA) expression have been reported in myelodysplastic syndrome (MDS). We aimed to investigate the unique patterns and prognostic significance of miRNA expression in Korean patients with MDS. METHODS: Bone marrow mononuclear cells were collected from eight healthy controls and 26 patients with MDS, and miRNAs were isolated and assessed via quantitative real-time polymerase chain reaction for selected miRNAs, including miR-21, miR-124a, miR-126, miR-146b-5p, miR-155, miR-182, miR-200c, miR-342-5p, miR-708, and Let-7a. RESULTS: MiR-124a, miR-155, miR-182, miR-200c, miR-342-5p, and Let-7a were significantly underexpressed in patients with MDS, compared to healthy controls. MiR-21, miR-126, 146b-5p, and miR-155 transcript levels were significantly lower in international prognostic scoring system lower (low and intermediate-1) risk MDS than in higher (intermediate-2 and high) risk MDS. Higher expression levels of miR-126 and miR-155 correlated with significantly shorter overall survival and leukemia-free survival. Higher miR-124a expression also tended to be related to shorter survivals. CONCLUSION: Although our study was limited by the relatively small number of patients included, we identified several miRNAs associated with pathogenesis, leukemic transformation, and prognosis in MDS.

Free radicals enzymatically triggered by Clonorchis sinensis excretory-secretory products cause NF-κB-mediated inflammation in human cholangiocarcinoma cells.

Chronic clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis worms and their excretory-secretory products, is associated with hepatobiliary damage, inflammation, periductal fibrosis and even development of cholangiocarcinoma. Our previous report revealed that intracellular reactive oxygen species were generated in C. sinensis excretory-secretory product-treated human cholangiocarcinoma cells; however, their endogenous sources and pathophysiological roles in host cells were not determined. In the present study, we found that treatment of human cholangiocarcinoma cells with excretory-secretory products triggered increases in free radicals via a time-dependent activation of NADPH oxidase, xanthine oxidase and inducible nitric oxide synthase. This increase in free radicals substantially promoted the degradation of cytosolic IκB-α, nuclear translocation of nuclear factor-κB subunits (RelA and p50), and increased κB consensus DNA-binding activity. Excretory-secretory product-induced nuclear factor-κB activation was markedly attenuated by preincubation with specific inhibitors of each free radical-producing enzyme or the antioxidant, N-acetylcysteine. Moreover, excretory-secretory products induced an increase in the mRNA and protein expression of the proinflammatory cytokines, IL-1ß and IL-6, in an nuclear factor-κB-dependent manner, indicating that enzymatic production of free radicals in ESP-treated cells participates in nuclear factor-κB-mediated inflammation. These findings provide new insights into the pathophysiological role of C. sinensis excretory-secretory products in host chronic inflammatory processes, which are initial events in hepatobiliary diseases.

PoCRIP1, Paralichthys olivaceus cysteine-rich intestinal protein 1: molecular characterization, expression analysis upon Edwardsiella tarda challenge and a possible role in the immune regulation.

Cysteine-rich intestinal protein (CRIP) is a LIM domain protein containing a zinc-finger motif and plays a role in the regulation of the inflammatory immune response. In the present study, we isolated a CRIP1 cDNA, designated PoCRIP1, from an olive flounder Paralichthys olivaceus intestine cDNA library by EST analysis. The PoCRIP cDNA consists of 421 bp with a polyadenylation signal sequence, AATAAA, and a poly(A) tail; it encodes a polypeptide of 76 amino acids containing a double zinc-finger motif (Cys(2)HisCys and Cys(4) sequences). The deduced amino acid sequence of PoCRIP1 showed 75.3-94.7% homology with CRIP1s of other species, including mammals. The PoCRIP1 transcript was highly expressed in the intestine and pyloric ceca and moderately expressed in the gill, heart, kidney, liver, muscle, spleen, skin, and stomach of normal conditioned flounder. Inducible expression of the PoCRIP1 transcript was observed in flounder challenged with Edwardsiella tarda, an economically important pathogen for aquaculture of flounder. Over-expression of PoCRIP1 augmented p65-driven flounder IL-6 promoter activity in HINAE cells. These results suggest that PoCRIP1 may function in the immune response of the flounder through the regulation of cytokine expression.

Cloning and functional characterization of the p65 subunit of NF-κB from olive flounder (Paralichthys olivaceus).

NF-κB is a master transcription factor found in almost all cell types that responds to diverse cellular stimuli by activating the expression of stress response genes, including immune-related genes. cDNA encoding the p65 subunit of olive flounder (Paralichthys olivaceus) NF-κB (Po-p65) was isolated through an EST analysis of an olive flounder cDNA library, a screen of BAC library, and rapid amplification of cDNA ends (RACE). The cDNA for Po-p65 encodes a polypeptide 626 amino acids in length containing a well-conserved Rel-homology domain (RHD). The primary sequence of Po-p65 showed strong homology with p65 from perch and zebrafish (82.7 and 64.4%, respectively), and shared 43.4-42.1% homology with p65 from other species, including mammals, while the N-terminal RHD of Po-p65 showed strong identity (95.6-67.8%) with that of other species. Po-p65 mRNA expression was detected in all flounder tissues examined. The over-expression of full-length Po-p65 (Po-p65f), but not of a Po-p65 C-terminus deletion mutant (Po-p65ΔC), stimulated κB element-driven reporter (κB-luc) activity in a dose-dependent manner and regulated the expression of p65 target genes, including TNF-α and IκB-α, in HINAE olive flounder cells. Po-p65f translocated to the nucleus following stimulation with poly I:C in HINAE cells. Together, these results suggest that Po-p65 is evolutionarily and functionally conserved in flounder and mammals and may provide clues to the detailed molecular mechanism(s) underlying immune response regulation in flounder.

Hypoxia induces the PDZ domain-containing syntenin in the marine teleost Paralichthys olivaceus.

Syntenin is a scaffolding PDZ domain-containing protein with diverse biological activities, including organization of protein complexes in the plasma membrane, regulation of B-cell development, intracellular trafficking, synaptic transmission, and cancer metastasis. In the present study, we isolated and characterized the cDNA of the olive flounder Paralichthys olivaceus syntenin, designated PoSyntenin. The full-length CDS of PoSyntenin with 5'- and 3'-UTR sequences is 2618bp long and consists of a 909bp open reading frame preceded by a 161bp 5'-UTR and followed by a 1551bp 3'-UTR. The PoSyntenin cDNA encodes a polypeptide of 302 amino acids containing two PDZ domains, which shares 61-80% homology with those of other species, including humans. Expression of the PoSyntenin mRNA was detectable from 1day post-hatching and constitutively in the brain, spleen, intestine, stomach, eye, liver, kidney, and gill of normal conditioned fish. Expression of the PoSyntenin mRNA was upregulated in the eye, liver, kidney, spleen, brain, gill, and intestine of flounder under hypoxia and was increased by treatment with the hypoxia-mimic CoCl(2) (a HIF-1 inducer) in HINAE cells. Taken together, these results suggest that PoSyntenin is a hypoxia target gene that has a potential role in the hypoxia response mechanism of fish.

Transcriptional induction of minichromosome maintenance protein 7 (Mcm7) in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

Clonorchiasis is an infection associated with bile duct malignancy and subsequent development of cholangiocarcinoma. This disease is mainly caused by Clonorchis sinensis worms and their excretory-secretory products (ESP). However, the precise molecular mechanisms of carcinogenesis remain to be determined. Previously, we established differential gene expression profiles from microarrays containing 23,920 human genes of known function in a human cholangiocarcinoma cell line, HuCCT1, treated with ESP. Among the upregulated genes, we focused on minichromosome maintenance protein 7 (Mcm7), which is implicated in various cancer types, and analyzed transcriptional regulation mediated by ESP to further elucidate its role in cholangiocarcinoma development. Global histone acetylation levels were increased in ESP-treated cells, along with histone acetyltransferase (HAT) protein expression. Detailed promoter analysis using reporter and chromatin immunoprecipitation assays revealed that transcriptional activation of Mcm7 is mediated by HAT recruitment to the promoter region upon C. sinensis ESP treatment. These findings contribute to clarification of the intrinsic mechanism underlying the cellular carcinogenesis process stimulated by Mcm7 in C. sinensis-treated host cells.

Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory-secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2-DE, quantitative image and MALDI-TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up-regulated and 34 down-regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox-regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells.

Differential gene expression profiling in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products.

Clonorchiasis is associated with bile duct malignancy and the subsequent development of cholangiocarcinoma. Although this is likely caused by adult Clonorchis sinensis and its excretory-secretory products (ESP), the precise molecular mechanisms remain obscure. To evaluate the effect of C. sinensis infection on differential gene expression in host hepatocytes, we examined the kinetics of changes in gene expression in the human cholangiocarcinoma cell line HuCCT1 treated with ESP at different times. Using complementary DNA microarrays containing 23,920 human genes of known function, we initially identified 435 genes altered by ESP treatment. Of these, 31 were up-regulated and 35 were down-regulated more than twofold in a time-dependent manner following ESP treatment. Clustering of these genes by function revealed that several were involved in the cell cycle, oncogenesis, protein modification, immunity, signal transduction, cell structure, and developmental processes. These findings should provide a fundamental basis for further analysis of the molecular pathways and mechanisms involved in C. sinensis infection of host cells.

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia.

Recent studies have reported that the cholinergic anti-inflammatory pathway regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X(7) receptor (P2X(7)R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fAbeta1-42)-induced ROS production by modulating ATP efflux-mediated Ca(2+) influx through P2X(7)R. Nicotine inhibited ROS generation in fAbeta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca(2+) influx in fAbeta(1-42)-stimulated microglia. Moreover, ATP release from fAbeta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca(2+) influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X(7)R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAbeta1-42-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X(7)R.

Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells.

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 microM) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-betaS that showed maximal IL-10 release were 100, 10, 100, and 100 microM respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP)=dATP>2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca(2+) release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down-regulated by either MRS2179 (a P2Y(1) antagonist) or 5'-AMPS (a P2Y(11) antagonist), indicating that both the P2Y(1) and P2Y(11) receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 microM) was restored by TNP-ATP (an antagonist of the P2X(1), P2X(3), and P2X(4) receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y(12) receptor antagonist) or pertussis toxin (PTX; a Gi protein inhibitor), indicating that the P2X(1), P2X(3), P2X(4)receptor group, or the P2Y(12) receptor, negatively modulate the P2Y(11) receptor or the P2Y(1) receptor, respectively.

ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in an autocrine fashion.

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta1-42) induced ATP release, which in turn activated NADPH oxidase via the P2X7 receptor (P2X7R). Reactive oxygen species (ROS) production in fAbeta1-42- treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X7R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta1-42-induced Ca2+ influx was mediated through P2X7R activation. In serial experiments, we found that microglial pretreatment with the P2X7R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 microM) or oxidized ATP (100 microM) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta1-42- stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.