Single-cell analysis identifies distinct CD4+ T cells associated with the pathobiology of pediatric obesity-related asthma.

Pediatric obesity-related asthma is characterized by non-atopic T helper 1 (Th1) inflammation and steroid resistance. CDC42 upregulation in CD4+T cells underliesTh1 inflammation but the CD4+T cell subtype(s) with CDC42 upregulation and their contribution to steroid resistance are not known. Compared to healthy-weight asthma, obesity-alone and healthy-weight controls, single-cell transcriptomics of obese asthma CD4+T cells revealed CDC42 upregulation in 3 clusters comprised of naïve and central memory T cells, which differed from the cluster enriched for Th1 responses that was comprised of effector T cells. NR3C1, coding for glucocorticoid receptor, was downregulated, while genes coding for NLRP3 inflammasome were upregulated, in clusters with CDC42 upregulation and Th1 responses. Conserved genes in these clusters correlated with pulmonary function deficits in obese asthma. These findings suggest that several distinct CD4+T cell subtypes are programmed in obese asthma for CDC42 upregulation, Th1 inflammation, and steroid resistance, and together contribute to obese asthma phenotype. Summary: CD4+T cells from obese children with asthma are distinctly programmed for non-allergic immune responses, steroid resistance and inflammasome activation, that underlie the obese asthma phenotype.

Crosstalk between CD4+ T Cells and Airway Smooth Muscle in Pediatric Obesity-related Asthma.

Rationale: Pediatric obesity-related asthma is a nonatopic asthma phenotype with high disease burden and few effective therapies. RhoGTPase upregulation in peripheral blood T helper (Th) cells is associated with the phenotype, but the mechanisms that underlie this association are not known. Objectives: To investigate the mechanisms by which upregulation of CDC42 (Cell Division Cycle 42), a RhoGTPase, in Th cells is associated with airway smooth muscle (ASM) biology. Methods: Chemotaxis of obese asthma and healthy-weight asthma Th cells, and their adhesion to obese and healthy-weight nonasthmatic ASM, was investigated. Transcriptomics and proteomics were used to determine the differential effect of obese and healthy-weight asthma Th cell adhesion to obese or healthy-weight ASM biology. Measurements and Main Results: Chemotaxis of obese asthma Th cells with CDC42 upregulation was resistant to CDC42 inhibition. Obese asthma Th cells were more adherent to obese ASM compared with healthy-weight asthma Th cells to healthy-weight ASM. Compared with coculture with healthy-weight ASM, obese asthma Th cell coculture with obese ASM was positively enriched for genes and proteins involved in actin cytoskeleton organization, transmembrane receptor protein kinase signaling, and cell mitosis, and negatively enriched for extracellular matrix organization. Targeted gene evaluation revealed upregulation of IFNG, TNF (tumor necrosis factor), and Cluster of Differentiation 247 (CD247) among Th cell genes, and of Ak strain transforming (AKT), Ras homolog family member A (RHOA), and CD38, with downregulation of PRKCA (Protein kinase C-alpha), among smooth muscle genes. Conclusions: Obese asthma Th cells have uninhibited chemotaxis and are more adherent to obese ASM, which is associated with upregulation of genes and proteins associated with smooth muscle proliferation and reciprocal nonatopic Th cell activation.

Toll-Like Receptor 7 Agonist RG7854 Mediates Therapeutic Efficacy and Seroconversion in Woodchucks With Chronic Hepatitis B.

Conventional treatment of chronic hepatitis B (CHB) is rarely curative due to the immunotolerant status of patients. RG7854 is an oral double prodrug of a toll-like receptor 7 (TLR7) agonist that is developed for the treatment of CHB. The therapeutic efficacy, host immune response, and safety of RG7854 were evaluated in the woodchuck model of CHB. Monotreatment with the two highest RG7854 doses and combination treatment with the highest RG7854 dose and entecavir (ETV) suppressed viral replication, led to loss of viral antigens, and induced seroconversion in responder woodchucks. Since viral suppression and high-titer antibodies persisted after treatment ended, this suggested that a sustained antiviral response (SVR) was induced by RG7854 in a subset of animals. The SVR rate, however, was comparable between both treatment regimens, suggesting that the addition of ETV did not enhance the therapeutic efficacy of RG7854 although it augmented the proliferation of blood cells in response to viral antigens and magnitude of antibody titers. The induction of interferon-stimulated genes in blood by RG7854/ETV combination treatment demonstrated on-target activation of TLR7. Together with the virus-specific blood cell proliferation and the transient elevations in liver enzymes and inflammation, this suggested that cytokine-mediated non-cytolytic and T-cell mediated cytolytic mechanisms contributed to the SVR, in addition to the virus-neutralizing effects by antibody-producing plasma cells. Both RG7854 regimens were not associated with treatment-limiting adverse effects but accompanied by dose-dependent, transient neutropenia and thrombocytopenia. The study concluded that finite, oral RG7854 treatment can induce a SVR in woodchucks that is based on the retrieval of antiviral innate and adaptive immune responses. This supports future investigation of the TLR7 agonist as an immunotherapeutic approach for achieving functional cure in patients with CHB.

Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks.

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients.

Treatment with the Immunomodulator AIC649 in Combination with Entecavir Produces Antiviral Efficacy in the Woodchuck Model of Chronic Hepatitis B.

As current interventions for chronic hepatitis B (CHB) rarely induce cure, more effective drugs are needed. Short-term treatment of woodchucks with the novel immunomodulator AIC649, a parapoxvirus-based stimulator of toll-like receptor 9 dependent and independent pathways, has been shown to reduce viral DNA and surface antigen via a unique, biphasic response pattern. The present study evaluated long-term AIC649 treatment in combination with Entecavir for potency and safety in woodchucks. AIC649 monotreatment induced modest reductions in serum viral DNA and surface and e antigens that were associated with the same biphasic response pattern previously observed. Entecavir monotreatment reduced transiently viremia but not antigenemia, while AIC649/Entecavir combination treatment mediated superior viral control. Undetectability of viral antigens and elicitation of antibodies in AIC649/Entecavir-treated woodchucks correlated with the expression of interferons and suppression of viral replication in liver. Combination treatment was well tolerated, and liver enzyme elevations were minor and transient. It was concluded that the AIC649-mediated effects were most likely based on an improvement and/or reconstitution of antiviral immune responses that are typically deficient in CHB. As a combination partner to Entecavir, the antiviral efficacy of AIC649 was markedly enhanced. This preclinical study supports future evaluation of AIC649 for treatment of human CHB.

Toll-Like Receptor 8 Agonist GS-9688 Induces Sustained Efficacy in the Woodchuck Model of Chronic Hepatitis B.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. APPROACH AND RESULTS: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. CONCLUSIONS: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.

Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon-α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus.

RG7834 is a small-molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon-α (wIFN-α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log10 from baseline and WHV DNA by a mean of 1.71 log10. ETV + wIFN-α reduced WHsAg and WHV DNA by means of 2.40 log10 and 6.70 log10, respectively. The combination of RG7834, ETV, and wIFN-α profoundly reduced WHsAg and WHV DNA levels by 5.00 log10 and 7.46 log10, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off-treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV-specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.

Liver-Targeted Toll-Like Receptor 7 Agonist Combined With Entecavir Promotes a Functional Cure in the Woodchuck Model of Hepatitis B Virus.

Current therapeutics for chronic infection with hepatitis B virus (HBV) rarely induce functional cure due to the immunotolerant status of patients. Small molecule agonists targeting toll-like receptor 7 (TLR7) have been shown to elicit a functional cure in animal models of HBV but sometimes with poor tolerability due to immune-related toxicities. In an effort to increase the therapeutic window of TLR7 agonists to treat chronic hepatitis B (CHB), we developed an oral TLR7 agonist, APR002, designed to act locally in the gastrointestinal tract and liver, thus minimizing systemic exposure and improving tolerability. Here, we describe the pharmacokinetic/pharmacodynamic (PK/PD) profile of APR002 in mice and uninfected woodchucks as well as the safety and antiviral efficacy in combination with entecavir (ETV) in woodchucks with CHB. Treatment of woodchucks chronically infected with woodchuck hepatitis virus (WHV) with weekly oral doses of APR002 was well-tolerated. While APR002 and ETV single agents did not elicit sustained viral control, combination therapy resulted in durable immune-mediated suppression of the chronic infection. These woodchucks also had detectable antibodies to viral antigens, enhanced interferon-stimulated gene expression, and loss of WHV covalently closed circular DNA. Conclusion: APR002 is a novel TLR7 agonist exhibiting a distinct PK/PD profile that in combination with ETV can safely attain a functional cure in woodchucks with chronic WHV infection. Our results support further investigation of liver-targeted TLR7 agonists in human CHB.

Antiviral Efficacy and Host Immune Response Induction during Sequential Treatment with SB 9200 Followed by Entecavir in Woodchucks.

SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) causing the induction of the interferon (IFN) signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV) versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated orally with SB 9200 (30 mg/kg/day) and ETV (0.5 mg/kg/day). Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-ß, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.

RNA binding by human Norovirus 3C-like proteases inhibits protease activity.

A highly active, fluorescence-based, in vitro assay for human Norovirus protease from genogroup I and II viruses was optimized utilizing as little as 0.25µM enzyme, pH 7.6, and substrate:enzyme of 50-100. Activity in Tris-HCl or sodium phosphate buffers was 2-fold less than HEPES, and 2-fold lower for buffer concentrations over 10mM. Protease activity at pH 7.6 was 73% (GI) or 63% (GII) of activity at the optimal pH 9.0. Sodium inhibited activity 2-3 fold, while potassium, calcium, magnesium, and manganese inhibited 5-10 fold. Differences in efficiency due to pH, buffer, and cations were due to changes in kcat and not Km. Norovirus protease bound short RNAs representing the 3' or 5' ends of the virus, inhibiting protease activity (IC50 3-5µM) in a non-competitive manner. Previous reports indicated participation of the protease in the Norovirus replicase complex. The current studies provide initial support for a defined role for the viral protease in Norovirus replication.

Mutations in HCV non-structural genes do not contribute to resistance to nitazoxanide in replicon-containing cells.

Nitazoxanide (NTZ) exhibits potent antiviral activity against hepatitis C virus (HCV) in cell culture. Previously, HCV replicon-containing cell lines resistant to NTZ were selected, but transfer the HCV NTZ-resistance phenotype was not observed following transfection of whole cell RNA. To further explore the nature of the resistance of HCV to NTZ, full length HCV replicon sequences were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines. Numerous nucleotide changes were observed in individual HCV genomes relative to the RP7 HCV consensus sequence, but no common mutations in the HCV non-structural genes or 3'-UTR were detected. A cluster of single nucleotide mutations was found within a 5-base portion of the 5'-UTR in 20/21 HCV replicon sequences from both resistant cell lines. Three mutations (5'-UTR G17A, G18A, C20U) were individually inserted into CON1 ('wild-type') HCV replicons, showed reduced replication (5 to 50-fold), but none conferred resistance to NTZ. RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under interferon. Transfection of cured NTZ-11 and TIZ-9 with either whole cell RNAs from RP7, NTZ-11, or TIZ-9, 'wild-type' or the 5'-UTR mutation-containing replicon RNAs exhibited an NTZ-resistance phenotype. TIZ (the active metabolite of NTZ) was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. These data confirm previous speculations that HCV resistance to NTZ is not due to mutations in the virus, and demonstrate that HCV resistance and most likely the antiviral activity of TIZ are due to interactions with cellular target(s).

Characterization of the West Nile virus protease substrate specificity and inhibitors.

West Nile virus (WNV), a mosquito-borne member of Flaviviridae, is a human pathogen causing widespread disease for which there is no vaccine or chemotherapy. The two-component viral serine protease consists of a heterodimeric complex between the hydrophilic domain of the cofactor, NS2B (NS2BH) and the protease domain (NS3-pro). The protease is essential for polyprotein processing followed by assembly of viral replicase and genome replication. Therefore, the protease is an excellent target for development of antiviral therapeutics. Here, we report the expression in Escherichia coli, purification, and characterization of biochemical and kinetic properties of the WNV protease. Furthermore, we show that the WNV and the dengue virus type 2 (DENV-2) proteases are inhibited by aprotinin with inhibitor constants of 0.16 and 0.026 microM, respectively. Molecular modeling of the WNV protease/aprotinin complex, based on the known crystal structures of the WNV NS2BH-N3pro and aprotinin, suggest a potentially strong interaction between the P2 Lys and the protease activator peptide, NS2BH. This conclusion based on molecular modeling is in agreement with our data of a higher k(cat)/Km value with the substrate, Boc-Gly-Lys-Arg-MCA than the Boc-Gly-Arg-Arg-MCA and is also consistent with the results of an earlier study that were based on substrate-based inhibitor peptides.

Hippocalcin increases phospholipase D2 expression through extracellular signal-regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin-induced phospholipase D2 expression.

We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal-regulated kinase (ERK)-mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN-PLD2) and hippocalcin co-transfected cells. DN-PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over-expression. Hippocalcin is a Ca2+-binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca2+, we investigated the possible role of hippocalcin in the LPA-induced elevation of intracellular Ca2+. When the intracellular Ca2+ level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin-induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI-PLC inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA-AM suggesting that activation of hippocalcin caused by increased intracellular Ca2+ is important to induce over-expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca2+, EGTA had little or no effect on the inhibition of hippocalcin-induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over-express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin-induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin-induced PLD2 expression, which is mediated by ERK activation.

Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase.

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.

Role of phospholipase D1 in neurite outgrowth of neural stem cells.

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.

De novo synthesis of negative-strand RNA by Dengue virus RNA-dependent RNA polymerase in vitro: nucleotide, primer, and template parameters.

By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40 degrees C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 micro M), the polymerase, and the template RNA. Even when the 3'-terminal sequence of template RNA was mutated from -GGUUCU-3' to -GGUUUU-3', a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3'-terminal dinucleotide CU of the template RNA indicated that change of the 3'-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3'-terminal nucleotide, from highest to lowest, is U, A - G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a "closed" form, catalyzing de novo initiation through synthesis of short primers, to an "open" form for elongation of a double-stranded template-primer.

Advanced lipid extraction method for the determination of the phospholipase D activity.

Phospholipase D is a ubiquitous enzyme that plays an important role in various lipid mediated cellular signaling pathways and produces rare phospholipids, phosphatidylethanol or phosphatidylbutanol, instead of phosphatidic acid with unique catalytic activity transphosphatidylation in the presence of primary alcohols. The reaction products, phosphatidylethanol or phosphatidylbutanol are used as markers of in vitro phospholipase D activity in many studies. For the sensitive detection of the phospholipase D products, we developed an advanced lipid extraction method that facilitates recovery of the compounds. With the new method, the activity change of phospholipase D by agonists could be detected more easily and the recovery rate was also increased. The increase of detected enzyme activity change was about double fold compared to the conventional lipid extraction method. This method provides selective force for the phospholipase D products in the extraction procedure.

Phosphatidylcholine-specific phospholipase C and RhoA are involved in the thyrotropin-induced activation of phospholipase D in FRTL-5 thyroid cells.

We investigated the possible involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in the thyroid-stimulating hormone (TSH)-induced protein kinase C (PKC)/phospholipase D (PLD) activation in FRTL-5 thyroid cells. Treatment of TSH resulted in both dose- and time-dependent increases in PLD activity. TSH induced translocations of PKCalpha and RhoA from the cytosol to the membrane within 30 min. TSH also stimulated diacylglycerol (DAG)/phosphorylcholine (PhoCho) production via PC-PLC. Pretreatment of the cells with D609, a potent inhibitor of PC-PLC, effectively inhibited the translocation of PKCalpha from the cytosol to the membrane and significantly decreased TSH-induced PLD activation. Moreover, C3 transferase, an inhibitor of RhoA, significantly inhibited PLD activity that was stimulated by TSH, which suggests that RhoA is also involved in TSH-induced PLD activation. As we expected, pretreatment of the cells with both C3 transferase and D609 completely inhibited the TSH-induced PLD activity. These findings suggest that DAG that is produced from cellular PC through PC-PLC plays an essential role in the TSH-induced PKC/PLD activation. Also, RhoA and PKCalpha are involved in the regulation of TSH-induced PLD activation in FRTL-5 thyroid cells.