5′-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 220.127.116.11) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in M. tuberculosis, was expressed in and purified from E. coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-L-homocysteine were poor substrates and no activity was detected with 5′-methylthioinosine, the other natural purines or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the Km value for MTA was 9.1 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicates that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
5′-methylthioadenosine phosphorylase; Rv0535; purine metabolism; Mycobacterium tuberculosis
A series of C-6 alkyl, cycloalkyl, and aryl-9-(β-d-ribofuranosyl)purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated. (Ph3P)4Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl)-purine (6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH3/MeOH gave the corresponding 6-alkyl-9-(β-d-ribofuranosyl) purine derivatives 7–11, respectively, in good yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl, and thienyl -9-(β-d-ribofuranosyl)purine derivatives 12–16, respectively in high yields. E. coli PNP showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12 and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In addition, 6-methyl-9-(β-d-arabinofuranosyl)purine (18) was prepared and evaluated.
purine nucleoside phosphorylase; organozinc halides; cross-coupling reactions; 6-alkyl; cycloalkyl/aryl/heterocyclylpurine ribonucleosides
Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2′-deoxyuridine to uracil and ribose 1-phosphate or 2′-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild type SpUP showed that substrate specificity is similar to that of EcUP, while EcUP is about sevenfold more efficient than SpUP. Biochemical studies on active site mutant SpUP showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.
Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia–telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.
Several lines of evidence support the view that autism is a typical member of a large family of immune-related, noninfectious, chronic diseases associated with postindustrial society. This family of diseases includes a wide range of inflammatory, allergic, and autoimmune diseases and results from consequences of genetic/culture mismatches which profoundly destabilize the immune system. Principle among these consequences is depletion of important components, particularly helminths, from the ecosystem of the human body, the human biome. Autism shares a wide range of features in common with this family of diseases, including the contribution of genetics/epigenetics, the identification of disease-inducing triggers, the apparent role of immunity in pathogenesis, high prevalence, complex etiologies and manifestations, and potentially some aspects of epidemiology. Fortunately, using available resources and technology, modern medicine has the potential to effectively reconstitute the human biome, thus treating or even avoiding altogether the consequences of genetic/cultural mismatches which underpin this entire family of disease. Thus, if indeed autism is an epidemic of postindustrial society associated with immune hypersensitivity, we can expect that the disease is readily preventable.
Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-d-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent Km (Km app) of 98 ± 6 μM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5′-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.
The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by over-expression of APRT. In vivo studies with 6-methylpurine-2′-deoxyriboside (MeP-dR), 2-F-2′-deoxyadenosine (F-dAdo), or 9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells co-expressing E. coli PNP did not enhance either the activation or the antitumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy and suggests ways to enhance the approach that are independent of APRT.
Adenine phosphoribosyltransferase; E. coli purine nucleoside phosphorylase; Fludarabine; 6-methylpurine-2′-deoxyriboside; 2-F-2′-deoxyadenosine; 9-β-D-arabinofuranosyl-2-fluoroadenine
Many solid tumors and hematologic malignancies lack expression of the enzyme methylthioadenosine phosphorylase (MTAP), due either to deletion of the MTAP gene or to methylation of the MTAP promoter. In cells that have MTAP, its natural substrate, methylthioadenosine (MTA), generated during polyamine biosynthesis, is cleaved to adenine and 5-methylthioribose-1-phosphate. The latter compound is further metabolized to methionine. Adenine and methionine are further metabolized and hence salvaged. In MTAP-deficient cells, however, MTA is not cleaved and the salvage pathway for adenine and methionine is absent. As a result, MTAP-deficient cells are more sensitive than MTAP-positive cells to inhibitors of de novo purine synthesis and to methionine deprivation. The challenge has been to take advantage of MTAP deficiency, and the changes in metabolism that follow, to design a strategy for targeted treatment. In this review, the frequency of MTAP-deficiency is presented and past and recent strategies to target such deficient cells are discussed, including one in which MTA is administered, followed by very high doses of a toxic purine or pyrimidine analog. In normal host cells, adenine, generated from MTA, blocks conversion of the analog to its toxic nucleotide. In MTAP-deficient tumor cells, conversion proceeds and the tumor cells are selectively killed. Successful mouse studies using this novel strategy were recently reported.
MTAP; MTA; adenine; 6-mercaptopurine; methotrexate
Gene transfer of the E. coli purine nucleoside phosphorylase (PNP) results in potent cytotoxicity after administration of the prodrug fludarabine phosphate (F-araAMP). Here we have tested whether application of this strategy in the context of replication-competent retrovirus (RCR) vectors, which can achieve highly efficient tumor-restricted transduction as well as persistent expression of transgenes, would result in effective tumor inhibition, or alternatively, would adversely affect viral replication. We found that RCR vectors could achieve high levels of PNP expression concomitant with the efficiency of their replicative spread, with significant cell killing activity in vitro and potent therapeutic effects in vivo. In U-87 xenograft models, replicative spread of the vector resulted in progressive transmission of the PNP transgene, as evidenced by increasing PNP enzyme activity with time following vector inoculation. Upon F-araAMP administration, high efficiency gene transfer of PNP by the RCR vector resulted in significant suppression of tumor growth and extended survival time. As the RCR mediates stable integration of the PNP gene and continuous expression, an additional round of F-araAMP administration resulted in further survival benefit. RCR-mediated PNP suicide gene therapy thus represents a highly efficient form of intracellular chemotherapy, and may achieve effective antitumor activity with less systemic toxicity.
retrovirus; purine nucleoside phosphorylase; suicide gene; oncolytic therapy; brain tumor
The crystal structure of purine nucleoside phosphorylase from grouper iridovirus was solved at 2.38 Å resolution.
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 Å resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 Å, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an α/β structure with a nine-stranded mixed β-barrel flanked by a total of nine α-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.
purine salvage; phosphorolysis; viral proteins; Tris-binding site
Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.
As part of an ongoing effort to develop new antiviral nucleoside analogs, our interest was drawn to N1-aryl purines as a novel structural class and potential scaffold for drug discovery. Herein, we describe the synthesis of N1-3-fluorophenyl-inosine (FPI) and N1-3-fluorophenyl-hypoxanthine (FP-Hx) and their antiviral activity against hantaviruses. The EC50 for FPI and FP-Hx were 94 μM and 234 μM, respectively, against Hantaan virus. FPI was not toxic to mammalian cells at concentrations that exhibited antiviral activity. Analysis of its metabolism revealed a low conversion of FPI in Vero E6 or human cells to a 5′-triphosphate, and it was a poor substrate for human purine nucleoside phosphorylase. Further, the compound did not alter GTP levels indicating FPI does not inhibit inosine monophosphate dehydrogenase. With respect to the virus, FPI did not decrease viral RNA levels or increase the mutation frequency of the viral RNA. This suggests that the antiviral activity of FPI might be solely due to the interaction of FPI or its metabolites with viral or host proteins involved in post-replication events that would affect the levels of infectious virus released. Synthesis of other compounds structurally similar to FPI is warranted to identify more potent agents that selectively abrogate production of infectious virus.
Hantaan virus; nucleosides; ribavirin; hantavirus
Recent revisions to the Sonsela Member of the Chinle Formation in Petrified Forest National Park have presented a three-part lithostratigraphic model based on unconventional correlations of sandstone beds. As a vertebrate faunal transition is recorded within this stratigraphic interval, these correlations, and the purported existence of a depositional hiatus (the Tr-4 unconformity) at about the same level, must be carefully re-examined.
Our investigations demonstrate the neglected necessity of walking out contacts and mapping when constructing lithostratigraphic models, and providing UTM coordinates and labeled photographs for all measured sections. We correct correlation errors within the Sonsela Member, demonstrate that there are multiple Flattops One sandstones, all of which are higher than the traditional Sonsela sandstone bed, that the Sonsela sandstone bed and Rainbow Forest Bed are equivalent, that the Rainbow Forest Bed is higher than the sandstones at the base of Blue Mesa and Agate Mesa, that strata formerly assigned to the Jim Camp Wash beds occur at two stratigraphic levels, and that there are multiple persistent silcrete horizons within the Sonsela Member.
We present a revised five-part model for the Sonsela Member. The units from lowest to highest are: the Camp Butte beds, Lot's Wife beds, Jasper Forest bed (the Sonsela sandstone)/Rainbow Forest Bed, Jim Camp Wash beds, and Martha's Butte beds (including the Flattops One sandstones). Although there are numerous degradational/aggradational cycles within the Chinle Formation, a single unconformable horizon within or at the base of the Sonsela Member that can be traced across the entire western United States (the “Tr-4 unconformity”) probably does not exist. The shift from relatively humid and poorly-drained to arid and well-drained climatic conditions began during deposition of the Sonsela Member (low in the Jim Camp Wash beds), well after the Carnian-Norian transition.
There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-β-ribosides and identified one with antiviral activity, 1-β-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC50 value of 10 and 4.4 μM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally-delivered ETAR offered protection to suckling mice challenged with HTNV with a ~25% survival at 12.5 and 25 mg/kg ETAR, and a MTD of 17.1 ± 0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5′-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.
Adenosine kinase (Ado kinase) from Mycobacterium tuberculosis is structurally and biochemically unique from other known Ado kinases. This purine salvage enzyme catalyzes the first step in the conversion of the adenosine analog, 2-methyl-Ado (methyl-Ado), into a metabolite with antitubercular activity. Methyl-Ado has provided proof of concept that the purine salvage pathway from M. tuberculosis may be utilized for the development of antitubercular compounds with novel mechanisms of action. In order to utilize this enzyme, it is necessary to understand the topography of the active site to rationally design compounds that are more potent and selective substrates for Ado kinase. A previous structure-activity relationship identified modifications to the base moiety of adenosine (Ado) that result in substrate and inhibitor activity. In an extension of that work, sixty-two Ado analogs with modifications to the ribofuranosyl moiety, modifications to the base and ribofuranosyl moiety, or modifications to the glycosidic bond position have been analyzed as substrates and inhibitors of M. tuberculosis Ado kinase. A subset of these compounds was further analyzed in human Ado kinase for the sake of comparison. Although no modifications to the ribose moiety resulted in compounds as active as Ado, the best substrates identified were carbocyclic-Ado, 8-aza-carbocyclic-Ado, and 9-[α-L-lyxofuranosyl]-adenine with 38%, 4.3%, and 3.8% of the activity of Ado respectively. The most potent inhibitor identified, 5′-amino-5′-deoxy-Ado, had a Ki = 0.8 μM and a competitive mode of inhibition. MIC studies demonstrated that poor substrates could still have potent antitubercular activity.
The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5′-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.
Although chronic aspiration has been associated with several pulmonary diseases, the inflammatory response has not been characterized. A novel rodent model of chronic aspiration was therefore developed in order to investigate the resulting innate immune response in the lung.
Gastric fluid or normal saline was instilled into the left lung of rats (n = 48) weekly for 4, 8, 12, or 16 weeks (n = 6 each group). Thereafter, bronchoalveolar lavage specimens were collected and cellular phenotypes and cytokine concentrations of IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha, and TGF-beta were determined.
Following the administration of gastric fluid but not normal saline, histologic specimens exhibited prominent evidence of giant cells, fibrosis, lymphocytic bronchiolitis, and obliterative bronchiolitis. Bronchoalveolar lavage specimens from the left (treated) lungs exhibited consistently higher macrophages and T cells with an increased CD4:CD8 T cell ratio after treatment with gastric fluid compared to normal saline. The concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha and TGF-beta were increased in bronchoalveolar lavage specimens following gastric fluid aspiration compared to normal saline.
This represents the first description of the pulmonary inflammatory response that results from chronic aspiration. Repetitive aspiration events can initiate an inflammatory response consisting of macrophages and T cells that is associated with increased TGF-beta, TNF-alpha, IL-1alpha, IL-1beta, IL-2 and fibrosis in the lung. Combined with the observation of gastric fluid-induced lymphocyitic bronchiolitis and obliterative bronchiolitis, these findings further support an association between chronic aspiration and pulmonary diseases, such as obliterative bronchiolitis, pulmonary fibrosis, and asthma.
Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure.
Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3121, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.
adenosine kinase; Mycobacterium tuberculosis
Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5′-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5′-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 μg/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 μg/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.
Cystic fibrosis is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, leading to altered ion transport, chronic infection, and excessive inflammation. Here we investigated regulation of CFTR in airway cell monolayers by adenosine, adenosine receptors, and arachidonic acid. Our studies demonstrate that the A2B adenosine receptor is expressed at high levels relative to the other adenosine receptor subtypes, with a characteristic low-affinity profile for adenosine-stimulated CFTR Cl− currents in both Calu-3 cells and CFBE41o- airway cell monolayers stably transduced with wild-type CFTR. The levels of adenosine found in sputum from patients with cystic fibrosis with moderate to severe lung disease stimulated apical prostaglandin release in Calu-3 and CFBE41o- cells, implicating adenosine regulation of phospholipase A2 (PLA2) activity. A2B adenosine receptor and arachidonic acid stimulation produced CFTR-dependent currents in airway monolayers and increased cAMP levels that were sensitive to cyclooxygenase inhibition. Arachidonic acid demonstrated dual regulation of CFTR, stimulating CFTR and Cl− currents in intact airway monolayers, and potently inhibiting PKA-activated Cl− currents in excised membrane patches. Cl− currents produced by arachidonic acid were sensitive to inhibition of PKA, cyclooxygenase, and 5-lipoxygenase. Together, the results provide a converging mechanism to link regulation of CFTR and airway cell inflammation through adenosine and adenosine receptors.
adenosine receptors; arachidonic acid; cystic fibrosis; inflammation; Phospholipase A2
A new discovery of skeletons of Revueltosaurus callenderi from the Upper Triassic Chinle Formation of Petrified Forest National Park, Arizona clearly shows that Revueltosaurus is not an ornithischian dinosaur as previously supposed. Features such as the presence of a postfrontal, crocodile-normal ankle and paramedian osteoderms with anterior bars place R. callenderi within the Pseudosuchia, closer to crocodylomorphs than to dinosaurs. Therefore, dental characters previously used to place Revueltosaurus within the Ornithischia evolved convergently among other archosaur taxa, and cannot be used to diagnose ornithischian dinosaur teeth. As a result, all other putative North American Late Triassic ornithischians, which are all based exclusively on teeth, are cast into doubt. The only reasonably well-confirmed Late Triassic ornithischians worldwide are Pisanosaurus mertii and an unnamed heterodontosaurid from Argentina. This considerably changes the understanding of early dinosaur diversity, distribution and evolution in the Late Triassic.
Revueltosaurus; Ornithischia; Pseudosuchia; Late Triassic; Petrified Forest National Park
The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to Escherichia coli is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the fimA gene. A minor component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We found that the aggregation of E. coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM α-CH3Man. The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in which E. coli was agglutinated by SIgA, the binding of SIgA to E. coli was not increased by the presence of the pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin.