M1 mRNA Research Articles

P.1.e.005 Possible involvement of p38MAPK and PP1 in impaired hippocampal function induced by 21-day propylthiouracil treatment

Hypothyroidism in the rat induced by perinatal exposure to propylthiouracil (PTU) is a useful animal model to study molecular changes underlying neurobehavioral defects associated with this condition. Understanding the developmental alterations in gene expression related to the neurobehavioral dysfunction should help to identify molecular markers for developmental neurotoxicity at an early stage of development.In the present study, we evaluate the effects of PTU on the expression of a set of genes implicated in neural network formation or synaptic function at a minimal dose of PTU causing behavioral alteration. Various doses of PTU were administered to dams from late pregnancy to the lactation period and the expression of selected genes in the hippocampus and the cerebral cortex of offspring was examined by quantitative RT-PCR. Behavioral performance of PTU-treated rats was also assessed. PTU-treated rats showed increased motor activity and impairment of E-maze learning at weaning and after maturation. At doses causing such behavioral alteration, expression of GAP-43 and M1 mRNAs was changed during neuronal network formation, suggesting that levels of these factors during development are important for accurate postnatal development and function.

P.1.e.004 Low tri-iodothyronine concentration is associated with greater depressive and anxiety symptoms in brain tumor patients

Hypothyroidism in the rat induced by perinatal exposure to propylthiouracil (PTU) is a useful animal model to study molecular changes underlying neurobehavioral defects associated with this condition. Understanding the developmental alterations in gene expression related to the neurobehavioral dysfunction should help to identify molecular markers for developmental neurotoxicity at an early stage of development.In the present study, we evaluate the effects of PTU on the expression of a set of genes implicated in neural network formation or synaptic function at a minimal dose of PTU causing behavioral alteration. Various doses of PTU were administered to dams from late pregnancy to the lactation period and the expression of selected genes in the hippocampus and the cerebral cortex of offspring was examined by quantitative RT-PCR. Behavioral performance of PTU-treated rats was also assessed. PTU-treated rats showed increased motor activity and impairment of E-maze learning at weaning and after maturation. At doses causing such behavioral alteration, expression of GAP-43 and M1 mRNAs was changed during neuronal network formation, suggesting that levels of these factors during development are important for accurate postnatal development and function.

SiRNA-loaded PEGylated porous silicon nanoparticles for lung cancer therapy

Biocompatible and biodegradable porous silicon nanoparticles (pSiNPs) with tunable pore size have been used as vehicles for drug delivery. Our pSiNPs have a mean size of 110 nm with an average pore size of 26 nm. Approximately 16.3 µg of siRNA can be loaded per mg of pSiNPs followed by polyethylene glycol (PEG) surface coating. siRNA-loaded PEGylated pSiNPs (P-pSiNPs) can be uptaken by A549 cells, and target pyruvate kinase isozyme type M2 (PKM2) mRNA and protein expression were effectively downregulated to 6.3 and 26.8 %, respectively. Cell viability decreased to 37.6 %; meanwhile, cell apoptosis upregulated to 17.3 %. The results prove that the siRNA-loaded pSiNPs coated with PEG can effectively deliver siRNA into cancer cells, and the efficiency is comparable to that of lipofectamine.

Secondary structure of a conserved domain in an intron of influenza A M1 mRNA.

Influenza A virus utilizes RNA throughout infection. Little is known, however, about the roles of RNA structures. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure starts 79 nucleotides downstream of the M2 mRNA 5′ splice site. Here, a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multibranch structure of this RNA. Imino proton NMR spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ∼14 °C higher than that for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture.

Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1–M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERβ, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33.M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups.Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus.These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERβ, but P4 may not regulate the expression of M3 and ERβ. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

The mammalian orthoreovirus bicistronic M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require interaction of 5′–3′ untranslated regions

Mammalian orthoreovirus mRNAs possess short 5′ UTR, lack 3′ poly(A) tails, and may lack 5′ cap structures at late times post-infection. As such, the mechanisms by which these viral mRNAs recruit ribosomes remain completely unknown. Toward addressing this question, we used bicistronic MRV M3 mRNA to analyze the role of 5′ and 3′ UTRs during MRV protein synthesis. The 5′ UTR was found to be dispensable for translation initiation; however, reducing its length promoted increased downstream initiation. Modifying start site Kozak context altered the ratio of upstream to downstream initiation, whereas mutations in the 3′ UTR did not. Moreover, an M3 mRNA lacking a 3′ UTR was able to rescue MRV infection to WT levels in an siRNA trans-complementation assay. Together, these data allow us to propose a model in which the MRV M3 mRNA initiates translation using a 5′ end-dependent, scanning mechanism that does not require the viral mRNA 3′ UTR or 5′–3′ UTRs interaction.

Expression of adrenergic and cholinergic receptors in murine renal intercalated cells.

ABSTRACTNeurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems.

Diagnostic value of alpha-fetoprotein, pyruvate kinase M2 and albumin mRNA expression in peripheral blood in hepatitis B virus-related cirrhosis

Diagnostic value of alpha-fetoprotein, pyruvate kinase M2 and albumin mRNA expression in peripheral blood in hepatitis B virus-related cirrhosis

Activated macrophages release microvesicles containing polarized M1 or M2 mRNAs

MVs are known vehicles of horizontal communication among cells, currently under scrutiny as powerful biomarkers in several pathological processes. The potential advantage of MVs relies on the assumption that their content reflects processes ongoing in pathologically relevant cell types. We have described that MVs of myeloid origin in the CSF are a marker of microglia/macrophage activation. Myeloid cells have different activation types, resulting in diverse functional phenotypes. Knowledge on the activation type of myeloid cells during disease would be of paramount importance for the understanding of ongoing pathogenic processes. We show here that macrophages activated in vitro in different ways all release increased amounts of MVs compared with NS cells. Moreover, we show that macrophage-derived MVs contain a repertoire of mRNAs that is not the result of casual sampling from the parental cells, as it is characterized by distinct mRNA enrichments and species. Nevertheless, mRNA content of MVs clearly allows identification in vivo of the activated phenotype of the cell of origin, indicating carryover of functional macrophage traits. We propose that detection of mRNAs in myeloid MVs permits identification of myeloid cell activation type during disease, allowing for further stratification of pathological processes.

Imaging of mRNA–Protein Interactions in Live Cells Using Novel mCherry Trimolecular Fluorescence Complementation Systems

Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA–protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA–protein interactions such as that between influenza viral protein NS1 and the 5’ UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA–protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA–protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.

DNA methylation status of NKX2-5, GATA4 and HAND1in patients with tetralogy of fallot

BackgroundNKX2-5, GATA4 and HAND1 are essential for heart development, however, little is known regarding their epigenetic regulation in the pathogenesis of tetralogy of fallot (TOF).MethodsMethylation levels were measured in three regions of NKX2-5 (M1: -1596 bp ~ -1374 bp, M2: -159 bp ~ 217 bp and M3: 1058 bp ~ 1524 bp), one region of GATA4 (M: -392 bp ~ 107 bp) and three regions of HAND1 (M1: -887 bp ~ -414 bp, M2: -436 bp ~ 2 bp and M3: 37 bp ~ 398 bp) using the Sequenom MassARRAY platform. QRT-PCR was used to analyze NKX2-5 and HAND1 mRNA levels in the right ventricular myocardium of TOF patients.ResultsTOF patients had a significantly higher NKX2-5_M3 median methylation level than controls (41.65% vs. 22.18%; p = 0.0074; interquartile range [IQR]: 30.46%–53.35%, N = 30 and 20.07%–24.31%, N = 5; respectively). The HAND1_M1 median methylation level was also significantly higher in TOF patients than controls (30.05% vs. 17.54%; p = 0.0054; IQR: 20.77%–40.89%, N = 30 and IQR: 14.69%–20.64%; N = 6; respectively). The methylation statuses of NKX2-5_M1, NKX2-5_M2, GATA4_M, HAND1_M2 or HAND1_M3 were not significantly different in TOF patients compared to controls. The methylation values for NKX2-5_M3 were negatively correlated with mRNA levels (r = - 0.463, p = 0.010, N = 30) and there was a significant association between HAND1_M1 methylation status and mRNA levels (r = - 0.524, p = 0.003, N = 30) in TOF patients.ConclusionsAberrant methylation statuses of the NKX2-5 gene body and HAND1 promoter regions are associated with the regulation of gene transcription in TOF patients and may play an important role in the pathogenesis of TOF.

An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers.

Forkhead box m1 mRNA expression of lung of preterm and term rabbits

Previous studies on forkhead box m1 (Foxm1) of mice demonstrated the correlation between this gene and lung maturation. However, no study has been conducted on human Foxm1 with regard to lung maturation. The aim of this study was to compare the mRNA expression of surfactant protein (SP)-A, -B, -C and Foxm1 gene of preterm rabbits to that of full-term ones and to determine the association between Foxm1 and lung maturation. New Zealand white rabbits were grouped according to gestational age. Cesarean sections were carried out after rabbits were divided into two groups of 30-31 days of gestation (term group, n=18) and 26-27 days of gestation (preterm group, n=18). mRNA expression levels of SP-A, -B, -C and Foxm1 were compared by using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The relative ratios of SP-A, -B and -C mRNA expression levels of the preterm to term groups were 0.380, 0.563 and 0.448:1, respectively, on qRT-PCR. By contrast, Foxm1 expression was increased in the preterm group and its relative expression ratio to the term group was 2.166:1 for RT-PCR and qRT-PCR, which was double that of the Foxm1 gene in the term group. Moreover, a significant correlation between the expressions of these genes was found. Foxm1 is considered to be an important gene required for the lung maturation of preterm rabbits in correlation with SP genes.

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.

Abstract 464: Tumor derived vacuolar ATPase subunit promotes M2 type polarization of macrophages.

Abstract Tumors and tumor associated macrophages (TAMs) express a unique surface vacuolar ATPase (V-ATPase) on the plasma membrane. In tumor cells, a 20 kd peptide is cleaved from a2 isoform of V-ATPase and is referred to as a2NTD; it is associated with in vitro induction of pro-tumorigenic properties in monocytes and IL-β secretion. In this study, we evaluated the immuno-modulatory properties of a2NTD in inducing murine peritoneal macrophage polarization and activation of inflammasome components both ex vivo and in vivo. Peritoneal macrophages from BALB/c mice were stimulated with recombinant a2NTD and the mRNA and protein expression of M1 (CD11c, TNF-α), M2 (Mannose receptor-1, Arginase-1, IL-10) markers, IL-1β and NLRP3 (inflammasome marker) were analyzed using quantitative real time PCR and flow cytometry. Further, mice were injected with a2NTD i.p. and peritoneal cells were analyzed for M1/M2 specific markers. The results of both in vivo and ex vivo analysis showed an elevated population of M2 subtype in a2NTD-treated mice compared to control (P<0.05). a2NTD upregulated the expression of IL-1β and NLRP3 in macrophages. These data demonstrated that a2NTD modulates macrophage polarization towards the M2 subtype functionally similar to tumor associated macrophages (TAMs). Since, tumor progression requires sterile inflammation and maturation of resident macrophages into TAMs; our data provide evidence that the surface expression of V-ATPase in tumor cells has a role in inducing the unique inflammatory environment of tumors by modulating macrophage phenotype and inducing IL-1β production through the action of a2NTD. The study also provides indication for the involvement of a2NTD in NLRP3 mediated inflammasome activation. Citation Format: Gajendra K. Katara, Mukesh K. Jaiswal, Alice Gilmam-Sacks, Kenneth D. Beaman. Tumor derived vacuolar ATPase subunit promotes M2 type polarization of macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 464. doi:10.1158/1538-7445.AM2013-464

Reduction of fatty acid oxidation and responses to hypoxia correlate with the progression of de-differentiation in HCC

The prognosis of patients with hepatocellular carcinoma (HCC) may be improved by novel treatments focusing on the characteristic metabolic changes of this disease. Therefore, we analyzed the biological interactions of metabolic features with the degree of tumor differentiation and the level of malignant potential in 41 patients with completely resectable HCC. The expression levels in resected samples of mRNAs encoded by genes related to tumor metabolism and metastasis were investigated, and the correlation between these expression levels and degrees of differentiation was analyzed. Of the 41 patients, 2 patients had grade I, 27 had grade II, and 12 had grade III tumors. Reductions in the levels of 3-hydroxyacyl-CoA dehydrogenase (HADHA) and acyl-CoA oxidase (ACOX)-2 mRNAs, and increases in pyruvate kinase isoenzyme type M2 (PKM2) mRNA were significantly correlated with the progression of de-differentiation. Analysis of partial correlation coefficients showed that the level of PKM2 mRNA expression was significantly correlated with those of pro-angiogenic genes, vascular endothelial growth factor (VEGF) and ETS-1. Moreover, the levels of VEGF-A and ETS-1 mRNA expression were independently correlated with that of the epithelial-mesenchymal transition (EMT)‑related gene SNAIL. These findings suggest that reductions in fatty acid oxidation and responses to hypoxia may affect the progression of malignant phenotypes in HCC.

Phase I Study of the Combination of Azacitidine (AZA) with MEC (Mitoxantrone, Etoposide and Cytarabine) Salvage Chemotherapy in Relapsed/Refractory Acute Myeloid Leukemia (AML): Early Results

AbstractAbstract 3616We recently reported that in addition to down-regulating DMNT1 mRNA and inducing DNA hypomethylation, AZA has the ability to reduce the mRNA and protein expression of M2, a subunit of Ribonucleotide Reductase (RNR) in AML cells [Blood 119(22): 5229–38, 2012]. RNR is an enzyme critical to DNA synthesis and repair that converts endogenous ribonucleotides to deoxynucleotides. Thus, we hypothesize that AZA-mediated downregulation of M2 results in decreased levels of endogenous triphosphate deoxynucleotides (dNTP). Thus, when AZA is combined with cytarabine (AraC), the backbone for several primary and salvage regimens in AML, the AraCTP/dNTP ratio would be expected to increase in favor of AraCTP DNA incorporation, thereby overcoming AraC resistance. Based on this rationale, we conducted a phase I trial of escalating doses of AZA (q24 h) on days 1–8 combined with unchanged mitoxantrone (6 mg/m2 q24h), etoposide (80 mg/m2 q24 h), and AraC (1 g/m2 q24 h) (MEC) salvage chemotherapy on days 3–8 in patients (pts) with relapsed or refractory AML. AZA doses were escalated (30, 50, or 75 mg/m2 q24h) with each dose level (DL) at which 3–6 pts were treated. Thirteen pts (39% male) with median age 52 (range 36–64) were enrolled. Five pts had primary refractory disease and 8 had relapsed AML (4 with 1st complete remission duration <12 months). Four pts had prior allogeneic stem cell transplant, and 4 pts had secondary AML (2 treatment related, 2 prior MDS). According to European LeukemiaNet AML Risk Groups, 2, 4, 4, and 3 pts had favorable, intermediate-I, intermediate-II, and adverse disease, respectively. One pt died on day 18 of sepsis, was not evaluable for response, and was replaced. Three pts are currently being treated on the last planned DL. In addition to anticipated myelosuppression, grade 3/4 non-hematologic adverse events at least possibly related to AZA included infections (n=10), diarrhea (n=5), increased bilirubin (n=1), increased AST/ALT (n=1), and vomiting (n=1). One pt (relapsed within 100 days of allogeneic transplant) did not recover platelets until day 58 after achieving morphologic complete remission (CR). Maximally tolerated dose has not been reached. CR and CR with incomplete count recovery (CRi) were achieved in 6/9 (67%; 5 CR and 1 CRi) pts who completed treatment. In responding pts, median time to ANC>500 was 35 days (range 23–45) and median time to platelets>50K was 35 days (range 22–45). Pharmacokinetics (PK) of AZA showed post-infusion biexponential decays with fast and slow plasma half-lives in the range of 10 minutes and 2 hours, respectively. Significant pharmacodynamic reduction in M2 mRNA and/or protein levels was observed in 3 pts analyzed, thus far. In conclusion, the combination of AZA with MEC salvage chemotherapy in advanced AML is well tolerated, not significantly different than MEC alone, and without prolongation in anticipated cytopenias. Early response rate and evidence of reduction in M2 as a proposed target of AZA are encouraging considering that most of the pts had high risk clinical and cytogenetic features. Updated clinical, PK and pharmacodynamic data will be presented. Supported by the National Cancer Institute of the National Institutes of Health Grant Number U01CA076576. Disclosures:Off Label Use: Azacitidine in AML.

A novel inhibitory mechanism of baicalein on influenza A/FM1/1/47 (H1N1) virus: interference with mid-late mRNA synthesis in cell culture

AimTo elucidate the inhibitory mechanism of baicalein on influenza A/FM1/1/47 (H1N1) virus in cell culture. MethodsThe inhibition of baicalein on influenza virus infection was investigated using a crystal violet staining assay. Three infection protocols were used to study the inhibitory effects of baicalein on different stages of the replication cycle of the influenza virus. Viral M1 mRNA expression was compared between drug-treated and untreated infected cells to determine the inhibitory effect of baicalein on viral mRNA synthesis using RT-PCR. ResultsBoth the A/FM1/1/47 (H1N1) and A/Beijing/32/92 (H3N2) influenza viruses were inhibited with different concentrations of baicalein in a dose-dependent manner. Treatment with baicalein after viral inoculation to MDCK cells resulted in stronger inhibitory effects compared with treatments prior to, and at the same time, as viral inoculation. Furthermore, no significant difference was observed between the viral M1 mRNA synthesis in the baicalein-treated group and the untreated group at 3 hpi (hours post-infection), compared to a clear decrease of viral mRNA synthesis at 6 and 9 hpi. ConclusionThis study has identified a novel inhibitory mechanism of baicalein on influenza A/FM1/1/47 (H1N1) virus via interference with mid-late mRNA synthesis in cell culture.

1807 The Comparison of Forkhead Box M1 MRNA Expression of Lung Tissues Between Preterm and term Rabbits

BackgroundRecent reports on Forkhead box m1 (Foxm1) of the mice provided correlations between this gene and lung maturation. However, there has been no study on human Foxm1 concerned with lung...