ABSTRACT
A functional renin-angiotensin system (RAS) is required for normal kidney development. Neonatal inhibition of the RAS in rats results in long-term pathological renal phenotype and causes hyaluronan (HA), which is involved in morphogenesis and inflammation, to accumulate. To elucidate the mechanisms, intrarenal HA content was followed during neonatal completion of nephrogenesis with or without angiotensin converting enzyme inhibition (ACEI) together with mRNA expression of hyaluronan synthases (HAS), hyaluronidases (Hyal), urinary hyaluronidase activity and cortical lymphatic vessels, which facilitate the drainage of HA from the tissue. In 6-8days old control rats cortical HA content was high and reduced by 93% on days 10-21, reaching adult low levels. Medullary HA content was high on days 6-8 and then reduced by 85% to 12-fold above cortical levels at day 21. In neonatally ACEI-treated rats the reduction in HA was abolished. Temporal expression of HAS2 corresponded with the reduction in HA content in the normal kidney. In ACEI-treated animals cortical HAS2 remained twice the expression of controls. Medullary Hyal1 increased in controls but decreased in ACEI-treated animals. Urine hyaluronidase activity decreased with time in control animals while in ACEI-treated animals it was initially 50% lower and did not change over time. Cells expressing the lymphatic endothelial mucoprotein podoplanin in ACEI-treated animals were increased 18-fold compared to controls suggesting compensation. In conclusion, the high renal HA content is rapidly reduced due to reduced HAS2 and increased Hyal1 mRNA expressions. Normal angiotensin II function is crucial for inducing these changes. Due to the extreme water-attracting and pro-inflammatory properties of HA, accumulation in the neonatally ACEI-treated kidneys may partly explain the pathological renal phenotype of the adult kidney, which include reduced urinary concentration ability and tubulointerstitial inflammation.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Glucuronosyltransferase/biosynthesis , Hyaluronic Acid/metabolism , Hyaluronoglucosaminidase/biosynthesis , Kidney/enzymology , Peptidyl-Dipeptidase A/metabolism , Animals , Enalapril/pharmacology , Gene Expression Profiling , Hyaluronan Synthases , Hyaluronoglucosaminidase/urine , Kidney Cortex/drug effects , Kidney Cortex/enzymology , Kidney Medulla/drug effects , Kidney Medulla/enzymology , Lymphatic Vessels/drug effects , Lymphatic Vessels/metabolism , Membrane Glycoproteins/biosynthesis , Organ Size , RNA, Messenger/biosynthesis , Rats , Rats, WistarABSTRACT
Influenza virus mRNA synthesis is primed by a capped oligonucleotide which is cleaved off from a cellular mRNA by a viral protein. The dinucleotide A3'p5'G can be used as a primer for the viral RNA polymerase mediated RNA synthesis in a cell-free system. Analogues of A3'p5'G have therefore been synthesized using the phosphotriester approach, and their priming ability for the influenza virus mRNA synthesis has been determined. An absence of the 2'-hydroxyl function in the guanosine residue in the dinucleotide, as in A3'p5'dG, drastically decreased its priming ability. Similarly, an alteration of the 3'----5' phosphate linkage to a 2'----5' phosphodiester linkage affected the priming ability quite severely. However a dinucleotide, with the 2'-hydroxyl function omitted in the adenosine moiety, as in dA3'p5'G, could still stimulate the mRNA synthesis. None of the modified dinucleotides inhibited A3'p5'G or globin mRNA primed influenza mRNA synthesis.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , DNA-Directed RNA Polymerases/metabolism , Dinucleoside Phosphates , Guanosine/analogs & derivatives , Influenza A virus/enzymology , Influenza B virus/enzymology , RNA, Messenger/biosynthesis , Adenosine Monophosphate/chemical synthesis , Animals , Cattle , Cell-Free System , Guanosine/chemical synthesis , Indicators and Reagents , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Foscarnet has previously been shown to inhibit influenza RNA polymerase activity. In this report the mode of inhibition of foscarnet has been investigated by enzyme-kinetic procedures and product analysis. Foscarnet shows noncompetitive inhibition with respect to ATP, CTP, and UTP, and a mixed inhibition with respect to GTP. In the presence of foscarnet the initiation of the mRNA synthesis can still occur, but the elongation is inhibited. The block of mRNA formation by foscarnet seems to occur after the synthesis of the 12-nucleotide-long conserved sequence found at the 5 prime end of the viral message.
Subject(s)
Antiviral Agents/toxicity , Influenza A virus/genetics , Organophosphorus Compounds/toxicity , Phosphonoacetic Acid/toxicity , RNA, Messenger/genetics , Transcription, Genetic/drug effects , Animals , DNA-Directed RNA Polymerases/metabolism , Foscarnet , Globins/genetics , Influenza A virus/drug effects , Kinetics , Phosphonoacetic Acid/analogs & derivatives , Phosphorus RadioisotopesABSTRACT
A rapid method for determining the concentration of ribonucleoside triphosphates in cell cultures is described. The pool sizes of the ribonucleoside triphosphates in uninfected MDCK cells were found to be, 0.28-0.59 mM CTP, 0.96-1.6 mM UTP, 6.5-7.8 mM ATP, and 1.1-1.4 mM GTP, depending on the condition of the cell-monolayer. Infection of MDCK cells with influenza A strains Victoria 3/75 x-47 or WSN resulted in insignificant changes in the ribonucleoside triphosphate concentrations. Treatment of infected and non-infected MDCK cells with ribavirin caused a decrease of 60 per cent in the GTP pool.
Subject(s)
Influenza A virus/physiology , Ribonucleotides/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Count , Cell Line , Cytidine Triphosphate/metabolism , Dogs , Guanosine Triphosphate/metabolism , Ribavirin/pharmacology , Uridine Triphosphate/metabolismABSTRACT
The effects of caps, dinucleotides, oligonucleotides and polynucleotides on influenza virus RNA polymerase activity have been investigated. The results show that both methyl groups in a cap are necessary for optimal stimulation of polymerase activity. Both m7G(5')ppp(5')Am and ApG stimulated the influenza RNA polymerase activity and seemed to interact at different sites. Out of the 16 homopolynucleotides tested, seven inhibited influenza RNA polymerase by 50% at 2-10 micrograms/ml. Poly(G) gave a 90% reduction of influenza virus plaque formation at 10 micrograms/ml. An oligodeoxyribonucleotide complementary to the 12 terminal nucleotides of the 3' end of influenza virus RNA was synthesized. This oligonucleotide did not selectively inhibit influenza RNA polymerase.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Guanine Nucleotides/pharmacology , Influenza A virus/enzymology , Nucleotides/pharmacology , Oligonucleotides/pharmacology , Polynucleotides/pharmacology , Globins/genetics , RNA, Messenger/biosynthesis , RNA, ViralABSTRACT
Analogues of pyrophosphate have been tested as inhibitors of influenza virus-RNA polymerase activity in cell-free assays. The most active compound, phosphonoformic acid (PFA), reduced the polymerase activity to 50 per cent at a concentration of 20 muM. The inhibition was dependent on the type of divalent cation present in the assay. PFA at a concentration of 400 muM also inhibited the influenza virus plaque formation by 90 per cent.
Subject(s)
DNA-Directed RNA Polymerases/antagonists & inhibitors , Diphosphates/pharmacology , Orthomyxoviridae/drug effects , Magnesium/pharmacology , Manganese/pharmacology , Orthomyxoviridae/enzymology , Orthomyxoviridae/growth & development , Structure-Activity Relationship , Viral Plaque AssayABSTRACT
Trisodium phosphonoformate selectively inhibits cell-free DNA polymerase activity induced by herpesvirus. The new inhibitor has an antiviral effect on herpes simplex virus types 1 and 2, pseudorables virus, and infectious bovine rhinotracheitis virus in cell culture. It has a good therapeutic activity against cutaneous herpes simplex virus infection in guinea pigs.
Subject(s)
Antiviral Agents , DNA-Directed RNA Polymerases/antagonists & inhibitors , Nucleic Acid Synthesis Inhibitors , Organophosphorus Compounds/pharmacology , Animals , Antiviral Agents/therapeutic use , Antiviral Agents/toxicity , Cell Line , Formates/pharmacology , Formates/toxicity , Guinea Pigs , Herpesviridae Infections/drug therapy , Organophosphorus Compounds/toxicity , Phosphonoacetic Acid/pharmacology , Simplexvirus/enzymologyABSTRACT
Ribavirin 5'-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5'-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5'-triphosphate and guanosine 5'-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP.
