MMLV Reverse Transcriptase Research Articles

Molecular markers for circulating tumor cells in breast cancer.

223 Background: Recently, we identified a six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8) for the RT-qPCR based detection of circulating tumor cells (CTC) in breast cancer patients. The aim of the present study was to evaluate the gene panel in further blood samples. Methods: Blood samples were taken from breast cancer patients with metastatic disease (MBC, N=10) or with no evidence of disease (NED, N=30). Putative CTC were enriched by Oncoquick density gradient centrifugation. Total RNA was isolated with RNeasy Micro Kit (QIAgen). Template cDNA was generated with M-MLV Reverse Transcriptase, RNase H Minus (Promega) and random nonamers as primers. RT-qPCR was performed in duplicate reactions using TaqMan Assays (Applied Biosystems) with default thermal cycling parameters. Raw data were analyzed with the AB7900 Sequence Detection Software version 2.2.2 using automatic baseline correction and manual cycle threshold setting. Gene expression was normalized to GAPDH expression. A threshold value TX for each gene X was set at two standard deviations above the mean dCtX value in the healthy control group. A patient was defined as CTC-positive, if at least one gene marker was over-expressed compared to the defined threshold. Results: The gene panel consisting of CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8 identified 4/11 MBC but only 5/27 NED patients as CTC positive (p=0.163). By adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the panel, 7/11 MBC but only 6/27 NED patients were CTC positive (p=0.018). The presence of CTC in NED patients correlated with pN staging (p=0.026). Only one out of the six CTC positive NED patients relapsed within the observation period (median 35 months, range 25-39 months from blood sampling). We observed no correlation of CTC positivity and recurrence in NED patients. Conclusions: The sensitivity of the RT-qPCR based CTC detection in breast cancer patients may be enhanced by adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8). Longer follow-up times are needed to evaluate the predictive value of the gene markers on survival.

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

Inhibition of HIV-1 and M-MLV reverse transcriptases by a major polyphenol (3,4,5 tri-O-galloylquinic acid) present in the leaves of the South African resurrection plant, Myrothamnus flabellifolia

A polyphenol-rich extract of the medicinal resurrection plant Myrothamnus flabellifolia was shown to inhibit viral (M-MLV and HIV-1) reverse transcriptases. Fractionation and purification of this extract yielded the major polyphenol, 3,4,5 tri-O-galloylquinic acid, as the main active compound. A sensitive, ethidium bromide based fluorescent assay, was developed and used to monitor the kinetics of M-MLV and HIV-1 reverse transcriptases in the presence and absence of 3,4,5 tri-O-galloylquinic acid. Kinetic monitoring of these enzymes in the presence of 3,4,5 tri-O-galloylquinic acid revealed non-competitive inhibition with IC50 values of 5 µM and 34 µM for the M-MLV and HIV-1 enzymes, respectively. We propose that 3,4,5 tri-O-galloylquinic acid and related polymers have potential as indigenous drugs for anti-viral therapy.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

The complete nucleotide sequences of bean common mosaic necrosis virus strains NL-5, NL-8 and TN-1

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) belong to the genus Potyvirus and are probably the most economically important viruses affecting common bean (Phaseolus vulgaris L.). Of the two viruses, BCMNV has been the most important in common bean production in North America, East Africa and Europe due to significant yield losses incurred from infected fields [10]. BCMV and BCMNV are transmitted in a non-persistent manner by several aphid species. All known strains of each virus can be highly seed-transmitted in bean and are distributed worldwide, in large part as a result of movement of contaminated seed [4, 5]. Drijfhout [4] originally separated the two viruses and their associated strains into eight different pathogroups based on host reaction to infection under specific temperature regimes. Strains NL-5 and NL-3 from Africa were originally assigned to the BCMV pathogroup VI by Drijfhout [4], and NL-8 was assigned to pathogroup III according to host responses in 12 different common bean host groups. The TN-1 strain from Tanzania was later assigned to pathogroup VI by Silbernagel [12]. Prior to 1992, all strains of BCMNV were designated as BCMV, at which time Vetten et al. [14] designated stains NL-3, NL-5 and NL-8 as serogroup A (BCMNV). All other known strains were assigned to serogroup B (BCMV) based on serological reactions to two highly specific monoclonal antibodies, and this was further supported by molecular analysis and ultrastructural comparisons [14]. Currently, several isolates of strain NL-3 have been sequenced and are available in GenBank; however, the sequences of strains NL-5, NL-8 and TN-1 are unavailable. Therefore, we report here the complete genome sequences of these three strains of BCMNV. Common bean seeds (variety ‘Dubbele Witte’) infected with NL-5, NL-8 and TN-1, respectively, were germinated in pots containing a mixture of peat moss and perlite (Sun Gro Horticulture Canada Ltd.) in the greenhouse, and plants were observed for symptoms. Tissues from symptomatic seedlings were evaluated for infection with BCMNV by direct ELISA using monoclonal antibodies specific for serogroup A, and also to serogroup B [11] to verify that there was no contamination with any strain of BCMV. Total nucleic acids were extracted from plant tissues, and cDNA was produced by reverse transcription using MMLV reverse transcriptase (Promega Corp., Madison, WI). Thermocycling parameters were optimized, and a final profile was employed that consisted of a single cycle of 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, and 3 min at 72 C; and a final extension for 7 min at 72 C. The initial reactions included oligo-dT24 as the reverse primer and a degenerate forward primer, 5’-GAA YAG CAA TGC NAT AG-3’, that produced a fragment approximately 3425 bp in length. The degenerate primer begins at nucleotide 6206, located in the NIa protein region. Subsequent reactions to complete the genome sequence were carried out using custom primer combinations based on the partial nucleotide sequence data obtained. The 5’-proximal region of the genomic sequence R. C. Larsen (&) USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350, USA e-mail: richard.larsen@ars.usda.gov

Antiretroviral activity of two polyisoprenylated acylphloroglucinols, 7-epi-nemorosone and plukenetione A, isolated from Caribbean propolis

Polyisoprenylated acylphloroglucinols have recently emerged as antitumoral agents. This study aims at elucidating the antiretroviral activity of two such compounds which were isolated from Caribbean propolis: 7-epi-nemorosone and plukenetione A, the structure of which is based on an adamantane moiety. Plukenetione A is for the first time shown to have antiretroviral activity. The isolation of both small molecules was carried out using RP-HPLC. Their antiretroviral activity was studied based on lentiviral particles produced in HEK293T cells from the SIV-based vector VLDBH; their cytotoxicity was monitored by MTT proliferation assay. The antiviral activity of 7-epi-nemorosone was studied in CEMx174-SEAP infected with the HIV-1-strain pNL4.3wt. Reverse transcriptase inhibition was determined by a standard two-step RT-PCR using MMLV RT. 7-epi-nemorosone and plukenetione A were found to be potent antilentiviral agents in the employed system, inhibiting viral infection at concentrations below 1 µM/2 µM, respectively. Whereas 7-epi-nemorosone was not able to inhibit the reverse transcriptase in vitro (IC50 > 25 µM), plukenetione A effectively inhibited its enzymatic activity at an IC50 of 1.75 µM. Despite 7-epi-nemorosone and plukenetione A sharing some structural core elements, the mechanism of action involved in their antiretroviral activity seems to be different. We propose that 7-epi-nemorosone inhibits the viral replication by interrupting the Akt/PKB signaling cascade, as was demonstrated previously in various cell lines. Since plukenetione A effectively inhibits the enzymatic activity of MMLV reverse transcriptase at concentrations that show antilentiviral activity, we suggest that this small molecule acts by interfering with the enzyme's catalytic site.

Interruption of cenph Causes Mitotic Failure and Embryonic Death, and Its Haploinsufficiency Suppresses Cancer in Zebrafish

Kinetochore proteins associate with centromeric DNA and spindle microtubules and play essential roles in chromosome segregation during mitosis. In this study, we uncovered a zebrafish mutant, stagnant and curly (stac), that carries the Tol2 transposon element inserted at the kinetochore protein H (cenph) locus. Mutant embryos exhibit discernible cell death as early as 20 hours postfertilization, extensive apoptosis, and upward curly tail during the pharyngula period and deform around 5 days postfertilization. The stac mutant phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph deficiency for stac mutants. We demonstrate that the intrinsic apoptosis pathway is hyperactivated in stac mutants and that p53 knockdown partially blocks excess apoptosis in stac mutants. Mitotic cells in stac mutants show chromosome missegregation and are usually arrested in G(2)/M phase. Furthermore, compared with wild type siblings, heterozygous stac fish develop invasive tumors at a dramatically reduced rate, suggesting a reduced cancer risk. Taken together, our findings uncover an essential role of cenph in mitosis and embryonic development and its association with tumor development.

Forced Expression of Methionine Adenosyltransferase 1A in Human Hepatoma Cells Suppresses in Vivo Tumorigenicity in Mice

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the principal methyl donor, and is encoded by MAT1A and MAT2A in mammals. Normal liver expresses MAT1A, which is silenced in hepatocellular carcinoma. We have shown that hepatoma cells overexpressing MAT1A grew slower, but whether this is also true in vivo remains unknown. To investigate the effect of overexpressing MAT1A on in vivo tumorigenesis, we generated stable transfectants of Huh7 cells overexpressing either MAT1A or empty vector. Real-time PCR and Western blotting were used to measure expression, and BALB/c nude mice were injected subcutaneously with untransfected or Huh7 cells transfected with empty or MAT1A expression vector to establish tumors. Tumor properties such as proliferation, angiogenesis, and apoptosis were compared, and microarray analysis was performed. Huh7 cells overexpressing MAT1A had higher S-adenosylmethionine levels but lower bromodeoxyuridine incorporation than control cells. Tumor growth rates and weights were lower in MAT1A transfected tumors. In addition, microvessel density and CD31 and Ki-67 staining were lower in MAT1A transfected tumors than control tumors, whereas the apoptosis index was higher in MAT1A-transfected tumors. Forced expression of MAT1A induced genes related to apoptosis and tumor suppression and lowered expression of cell growth and angiogenesis proteins. Our data demonstrate in vivo overexpression of MAT1A in liver cancer cells can suppress tumor growth. They also suggest inducing MAT1A expression might be a strategy to treat hepatocellular carcinoma.

Expression and Function of Epithelial Anoctamins

The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1-ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca(2+)-activated Cl(-) conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl(-) conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca(2+) sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca(2+)-dependent Cl(-) channels.

Quantitation of WT1 Transcripts by Copy Number Provides a Sensitive Indication of Minimal Residual Disease (MRD) and Response to Adoptive Cell Therapy in Patients Transplanted for Ph(+) CML.

Abstract 4489▪The Wilm's tumor gene, WT-1, is markedly overexpressed in a high proportion of acute leukemias, advanced MDS and Ph(+) CML. Prior studies, employing semiquantitative PCR-based techniques, have suggested that elevated levels of WT-1 transcripts in bone marrow and PBMC are indicative of MRD, and predictive of relapse in AML and CML following chemotherapy. We have now developed a quantitative real time PCR-based assay for WT-1 transcripts and used it to evaluate the long term effects of donor lymphocyte infusions in a series of patients with Ph(+) CML treated for relapse or persistent MRD following allogeneic marrow transplants between 1993 and 2004 who are currently in molecular or cytogenetic remission.To quantitate WT-1 mRNA copy number in the marrow and PBMC, total RNA from patient samples received at different times that had been extracted with trizol reagent and stored at -20°C was transcribed into cDNA using MMLV reverse transcriptase. Real time PCR was performed with 1-2 μg of total RNA using the ABI 7300 instrument with a 25 base pair FAM labeled probe containing a sequence located at the boundary of exon 6 and 7 of the WT-1 gene in order to eliminate amplification of genomic DNA and insure measurement of all transcribed WT-1 isoforms. The forward and reverse primers were synthesized from sequences contained in exon 6 and 7 of the WT-1 gene respectively. Cloned plasmids containing defined copy numbers of WT-1 and human GAPDH were used to prepare standard curves using serial dilutions. Quantitation of WT-1 copy number expression was performed by calibration against these standard curves. The expression of WT-1 was normalized against the expression of the control GAPDH to adjust for variation in RNA and cDNA synthesis.Using this assay, we evaluated sequential samples from each patient for correlations between WT-1 copy number detected and the results of qualitative PCR assays for bcr/abl fusion gene transcripts previously performed on the same samples using nested primers as well as the results of clinical, hematologic and cytogenetic analyses of each patient obtained at each time of sampling. Blood samples from a group of 8 normal volunteers served as negative controls.The average WT-1 copy number in the blood of normal volunteers was 25 copies/ μg RNA (range 2-60). With one exception, blood and marrow samples from patients with CML in molecular remission (bcr/abl not detected) had WT-1 copy numbers < 50 copies/μg RNA. One sample of marrow from a patient who had been bcr/abl + until 6 months post transplant and had WT-1 copy numbers that had fallen from 12,932 one month post transplant to 1855 at that time, became bcr/abl negative at 11 months but still had a WT-1 copy number of 931/ μg RNA. In contrast, patients with molecular disease (+ bcr/abl) maintained WT-1 copy numbers ranging from 70-1000/ μg RNA. Samples from patients with cytogenetic relapse consistently had WT-1 copy numbers exceeding 1000/ μg RNA, with levels exceeding 10,000 in 4/6 such patients tested. In a subset of patients who have failed to achieve or sustain molecular remission following DLI, marrow and blood samples with detectable bcr/abl transcripts and WT-1 copy numbers ranging from 60-3000 have been repeatedly detected for periods of up to 6 years without additional treatment and without disease progression implying an ongoing mechanism of disease control. Therefore, quantitation of WT-1 copy number provides a useful technique for monitoring minimal residual disease and may be helpful in identification of patients at higher risk of cytogenetic relapse. Disclosures:No relevant conflicts of interest to declare.

Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( K d = 9.019 μM)), RNase A ( K d = 2.33 μM) reverse transcriptase ( K d = 0.255 μM) and Taq polymerase ( K d = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.

Activin-Like Kinase 5 (ALK5) Mediates Abnormal Proliferation of Vascular Smooth Muscle Cells from Patients with Familial Pulmonary Arterial Hypertension and Is Involved in the Progression of Experimental Pulmonary Arterial Hypertension Induced by Monocrotaline

Mutations in the gene for the transforming growth factor (TGF)-beta superfamily receptor, bone morphogenetic protein receptor II, underlie heritable forms of pulmonary arterial hypertension (PAH). Aberrant signaling via TGF-beta receptor I/activin receptor-like kinase 5 may be important for both the development and progression of PAH. We investigated the therapeutic potential of a well-characterized and potent activin receptor-like kinase 5 inhibitor, SB525334 [6-(2-tert-butyl-5-{6-methyl-pyridin-2-yl}-1H-imidazol-4-yl)-quinoxaline] for the treatment of PAH. In this study, we demonstrate that pulmonary artery smooth muscle cells from patients with familial forms of idiopathic PAH exhibit heightened sensitivity to TGF-beta1 in vitro, which can be attenuated after the administration of SB525334. We further demonstrate that SB525334 significantly reverses pulmonary arterial pressure and inhibits right ventricular hypertrophy in a rat model of PAH. Immunohistochemical studies confirmed a significant reduction in pulmonary arteriole muscularization induced by monocrotaline (used experimentally to induce PAH) after treatment of rats with SB525334. Collectively, these data are consistent with a role for the activin receptor-like kinase 5 in the progression of idiopathic PAH and imply that strategies to inhibit activin receptor-like kinase 5 signaling may have therapeutic benefit.

Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.

Seasonal Variation in Gene Expression of Bovine Oocytes and the Resultant Embryos.

Developmental competence of bovine oocytes is known to decrease during the hot season. The mechanism underlying this disruption, however, is not entirely clear. The present study examined seasonal effects on the expression of genes involved in oocyte maturation, zygote formation and early embryonic development. Bovine oocytes were aspirated from ovaries collected from a local slaughterhouse during the summer (Jun-Sep) and winter (Dec-May). Oocytes were matured (22 h), in-vitro fertilized (18 h, 38.5°C, 5% CO2) and cultured (KSOM, 38.5°C, 5% CO2, 5% O2) for 8 days. In each season (× four replicates), samples (n = 20) of GV- and MII-oocytes and zygotes, and of preimplantation embryos at the 2-, 4- and 8-cell and blastocyst stages (n = 10, 5, 3 and 4, respectively) were collected and total RNA was isolated, using 500 µl Trizol reagent and 10 µl glycogen. cDNA was generated from each sample using M-MLV reverse transcriptase (for 1 h at 42°C) and qPCR was carried out with primers for GDF9, POU5F1 (previously known as OCT4) and C-MOS using 18S and GAPDH as reference genes. Unexpectedly, gene expression of 18S was stable throughout all developmental stages in both seasons, while that of GAPDH varied among and between seasons, with decreases in MII-oocytes and 8-cell-stage embryos during the summer. Thus, gene expression was referenced to the former gene. The average cleavage rate (2- to 4-cell stage, 44 h post fertilization) did not differ between seasons (80 ± 10.5 vs. 73 ± 9.2% for winter and summer, respectively) however, the proportion of 2-cell stage in the summer was twofold higher from that of 4-cell. The percentage of embryos that developed to the blastocyst stage was higher in the winter than in the summer (23 ± 2.3 vs. 9 ± 3.5 %, respectively; P<0.05). In GV oocytes and putative zygotes, expression of all the examined genes was stable and did not differ between seasons. On the other hand at the MII stage, the expression of all genes decreased in the summer, most markedly that of C-MOS and POU5F1. In 2-cell-stage embryos, expression of all the examined genes was unstable among and between seasons. At the 4-cell stage, opposite patterns were noted between seasons, with decreased expression of GDF9 in the winter and of POU5F1 in the summer. Similarly, the expression of POU5F1 in 8-cell-stage embryos and blastocysts was lower in the summer than in the winter. Taken together, results indicated seasonal variations in gene expression in oocytes and their resultant embryos, which might explain, at least in part, the mechanism underlying the disruption of oocyte developmental competence during the hot season.

Kinetic analysis of template competitive inhibitors of reverse transcriptases from HIV‐1, MMLV, and AMV

Many viral diseases threaten people's health and there are too few vaccines or viral inhibitors to fight against these viruses. One example is the HIV retrovirus. HIV encodes the enzyme reverse transcriptase (RT) that converts single‐stranded RNA genome of the retrovirus into double‐stranded DNA before the cell begins viral replication. The goal of our research has been to synthesize nucleotide inhibitors that can directly inhibit the activity of RT and prevent viral replication. Four inhibitors molecules synthesized and characterized kinetically are: 1) 2‐S‐(3,4‐dichlorobenzyl)‐etheno‐dATP, 2) 2‐S‐(4‐chlorobenzyl)‐etheno‐dATP, 3) 2‐S‐benzyl‐etheno‐dATP, and 4) 2‐S‐(4‐methoxybenzyl)‐etheno‐dATP. Five component inhibition assays were performed, either as IC50 or Ki plots, against three enzymes, HIV‐1, MMLV, and AMV reverse transcriptases. The results of enzyme assays performed to test the inhibition of HIV RT will be presented.

Genes Proximal and Distal to MYCN Are Highly Expressed in Human Neuroblastoma as Visualized by Comparative Expressed Sequence Hybridization

MYCN amplification is associated with poor prognosis in neuroblastoma disease. To improve our understanding of the influence of the MYCN amplicon and its corresponding expression, we investigated the 2p expression pattern of MYCN amplified (n = 13) and nonamplified (n = 4) cell lines and corresponding primary tumors (n = 3) using the comparative expressed sequence hybridization technique. All but one MYCN amplified cell line displayed overexpression at 2p. Expression peaks were observed frequently at 2pter and less frequently at 2p24 (MYCN locus), 2p23.3-23.2, and/or 2p23.1. Importantly, cell lines and two corresponding primary tumors displayed expression peaks at similar loci. No significant 2p24 expression level was observed for those cell lines displaying a low amplification rate (n = 3) by comparative genomic hybridization. Only the cell lines with an enhanced peak at 2p23.2-23.3 displayed coamplification of the ALK gene (2p23.2), reported to be associated with unfavorable prognosis. Finally, two of four cell lines without MYCN amplification, both derived from patients with poor outcome, also showed an expression peak at 2p23.2. These data indicate that, besides MYCN, other genes proximal and distal to MYCN are highly expressed in neuroblastoma. The prognostic significance of expression peaks at 2p23.2-23.3, independent of MYCN and ALK status, remains to be investigated.

Leptin-Dependent Toll-Like Receptor Expression and Responsiveness in Preadipocytes and Adipocytes

Leptin, an adipokine mainly produced by adipocytes, has been well characterized with regard to its regulatory function on immune cells. Thus the question occurred of how adipocytes and preadipocytes interact with the immune system and whether or not this communication is regulated by leptin. With the present study we evaluated the Toll-like receptor (TLR) expression and TLR ligand-specific activation of murine preadipocytes and adipocytes in the presence [wild type (WT), 3T3L1] or absence of leptin (ob/ob) or leptin signaling (db/db). The ob/ob as well as db/db adipocytes and preadipocytes were characterized by a significant up-regulation of TLR1 to -9 expression when compared with WT cells. In WT preadipocytes the TLR responsiveness increased during maturation to adipocytes; however, stimulation of ob/ob and db/db cells resulted in a 10- to 20-fold higher interleukin-6 production. Signaling studies revealed, in addition to the increased TLR expression, alterations in the phosphoinositide 3 kinase signaling cascade in ob/ob and db/db cells as an explanation for this increased responsiveness. In conclusion, the present study indicates the expression and responsiveness of TLR1 to -9 in murine preadipocytes as well as adipocytes, both of which are strongly regulated by the adipokine leptin. In summary, these data further emphasize the role of fat tissue in the immune system.