The RimL transacetylase provides resistance to translation inhibitor microcin C.

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.

Electrophysiological Profile of Different Antiviral Therapies in a Rabbit Whole-Heart Model.

Antiviral therapies for treatment of COVID-19 may be associated with significant proarrhythmic potential. In the present study, the potential cardiotoxic side effects of these therapies were evaluated using a Langendorff model of the isolated rabbit heart. 51 hearts of female rabbits were retrogradely perfused, employing a Langendorff-setup. Eight catheters were placed endo- and epicardially to perform an electrophysiology study, thus obtaining cycle length-dependent action potential duration at 90% of repolarization (APD90), QT intervals and dispersion of repolarization. After generating baseline data, the hearts were assigned to four groups: In group 1 (HXC), hearts were treated with 1 µM hydroxychloroquine. Thereafter, 3 µM hydroxychloroquine were infused additionally. Group 2 (HXC + AZI) was perfused with 3 µM hydroxychloroquine followed by 150 µM azithromycin. In group 3 (LOP) the hearts were perfused with 3 µM lopinavir followed by 5 µM and 10 µM lopinavir. Group 4 (REM) was perfused with 1 µM remdesivir followed by 5 µM and 10 µM remdesivir. Hydroxychloroquine- and azithromycin-based therapies have a significant proarrhythmic potential mediated by action potential prolongation and an increase in dispersion. Lopinavir and remdesivir showed overall significantly less pronounced changes in electrophysiology. In accordance with the reported bradycardic events under remdesivir, it significantly reduced the rate of the ventricular escape rhythm.

Revealing the Inhibition Mechanism of RNA-Dependent RNA Polymerase (RdRp) of SARS-CoV-2 by Remdesivir and Nucleotide Analogues: A Molecular Dynamics Simulation Study.

Antiviral drug therapy against SARS-CoV-2 is not yet established and posing a serious global health issue. Remdesivir is the first antiviral compound approved by the US FDA for the SARS-CoV-2 treatment for emergency use, targeting RNA-dependent RNA polymerase (RdRp) enzyme. In this work, we have examined the action of remdesivir and other two ligands screened from the library of nucleotide analogues using docking and molecular dynamics (MD) simulation studies. The MD simulations have been performed for all the ligand-bound RdRp complexes for the 30 ns time scale. This is one of the earlier reports to perform the MD simulations studies using the SARS-CoV-2 RdRp crystal structure (PDB ID 7BTF). The MD trajectories were analyzed and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations were performed to calculate the binding free energy. The binding energy data reveal that compound-17 (-59.6 kcal/mol) binds more strongly as compared to compound-8 (-46.3 kcal/mol) and remdesivir (-29.7 kcal/mol) with RdRp. The detailed analysis of trajectories shows that the remdesivir binds in the catalytic site and forms a hydrogen bond with the catalytic residues from 0 to 0.46 ns. Compound-8 binds in the catalytic site but does not form direct hydrogen bonds with catalytic residues. Compound-17 showed the formation of hydrogen bonds with catalytic residues throughout the simulation process. The MD simulation results such as hydrogen bonding, the center of mass distance analysis, snapshots at a different time interval, and binding energy suggest that compound-17 binds strongly with RdRp of SARS-CoV-2 and has the potential to develop as a new antiviral against COVID-19. Further, the frontier molecular orbital analysis and molecular electrostatic potential (MESP) iso-surface analysis using DFT calculations shed light on the superior binding of compound-17 with RdRp compared to remdesivir and compound-8. The computed as well as the experimentally reported pharmacokinetics and toxicity parameters of compound-17 is encouraging and therefore can be one of the potential candidates for the treatment of COVID-19.

Effects of cadmium chloride inhalation on airflow limitation to histamine, carbachol and adenosine 5'-monophosphate assessed by barometric whole body plethysmography in healthy dogs.

The effects of pharmacological bronchoprovocation on airflow patterns and surrogate respiratory parameters assessed by barometric whole body plethysmography (BWBP) were investigated in healthy dogs, previously exposed to cadmium chloride inhalation. BWBP-derived respiratory variables were calculated (1) at baseline and (2) following nebulisation of increasing concentrations of histamine, carbachol and adenosine 5'-monophosphate (AMP) until enhanced pause (PENH) increased to 300% of baseline (PCPENH300). Bronchoalveolar lavage fluid (BALF) cytology before (BCC) and after (ACC) cadmium chloride inhalation revealed cadmium-induced airway inflammation. Neutrophils increased from 6.7 +/- 7.3% (728 +/- 104/microL) BCC to 77.8 +/- 8.6% (3255 +/- 1407/microL) ACC. PCPENH300 for all three agonists significantly decreased ACC (means+/-SD) as follows: PCPENH300(histamine) 0.72 +/- 0.28 mg/mL BCC, and 0.35 +/- 0.31 mg/mL ACC (P<0.02); PCPENH300(carbachol) 0.34 +/- 0.16 mg/mL BCC, and 0.064 +/- 0.032 mg/mL ACC (P<0.02); PCPENH300(AMP) 1000 mg/mL BCC, and 415 +/- 398 mg/mL ACC (P<0.03). The only clinical sign shown was coughing. It was concluded that airway hyperresponsiveness after induced airway inflammation can be determined by BWBP in conscious small sized dogs. BWBP could be a suitable technique to study the respiratory effects of urban environmental pollution in pets.

Regulation of Expression of Hyperalgesic Priming by Estrogen Receptor α in the Rat.

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N6-cyclopentiladenosine still produced hyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming. PERSPECTIVE: This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Growth inhibition of human tumor cells in soft-agar cultures by treatment with low levels of adenosine 5'-triphosphate.

Treatment of a variety of human tumor cells in monolayer cultures with low levels (40 to 80 microM) of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) was recently shown to produce arrest of cellular growth in the S phase of the cell cycle (E. Rapaport, J. Cell. Physiol., 114: 279-283, 1983). We now demonstrate that exposure of two well-characterized colonic adenocarcinoma (HT-29 and SW-620) and two pancreatic adenocarcinoma (CAPAN-1 and PANC-1) cell lines in soft-agar cultures to exogenously supplied 5 to 20 microM ATP results in substantial inhibition of cellular growth. Exposure of the cells to 5 to 20 microM ADP produces slightly smaller growth-inhibitory effects, while 20 microM adenosine 5'-monophosphate or adenosine have marginal effects on cellular proliferation in these systems. Successful demonstration of these effects requires the use of heat-inactivated fetal bovine serum, since normal fetal bovine serum possesses enzymatic activities which catalyze the rapid degradation of adenine nucleotides. Tumor cell growth was assayed by the well-established colony formation assay as well as by [3H]thymidine incorporation into acid-insoluble material. [3H]Thymidine incorporation is performed 4 to 14 days after plating and correlates well with results obtained by colony formation assays. Due to the ectoenzymatic activities of the cells which include adenosinetriphosphatase and adenosinediphosphatase catalyzing the dephosphorylation of ATP and ADP, the effective levels of ATP that inhibit the growth of human tumor cells in this system, which is widely claimed to predict the in vivo response of a tumor, are lower than the 5 to 20 microM which are exogenously supplied. The two previously characterized, well-differentiated pancreatic and colonic tumor cell lines (CAPAN-1 and HT-29) were shown to exhibit higher chemosensitivity towards treatment with ATP and ADP than did the lesser-differentiated pancreatic and colonic tumor cell lines (PANC-1 and SW-620).

Radioprotection of mouse hemopoiesis by dipyridamole and adenosine monophosphate in fractionated treatment.

The purpose of the studies reported here was to investigate the ability of the combined administration of dipyridamole and adenosine monophosphate, drugs known to elevate extracellular adenosine, to protect mice undergoing treatment with fractionated irradiation (five doses of 2 or 3 Gy each) given at 24-h intervals. Based on observations of hemopoietic recovery (endogenous hemopoietic spleen colony formation, marrow granulocyte-macrophage colony-forming cells, peripheral blood cells) after the completion of fractionated irradiation and on survival studies, it was demonstrated that the repeated administration of the drugs 60 min before each of the radiation fractions mitigates the hemopoietic injury and enhances the survival of mice irradiated with an additional "top-up" dose. It could be deduced that the single protective actions of the drugs retain their efficacy in repeated treatment and enhance the sparing effect of dose fractionation on hemopoiesis. Interestingly, the toxic side effects of the drugs tend to decrease when they are administered repeatedly, probably due to the development of tolerance to their cardiovascular action. This reduction in toxicity offers benefit with respect to the potential use of these hemopoiesis-protecting drugs in clinical radiotherapy.

In vivo testing of Enterogenin for a mutagenic activity of bone marrow hemopoietic tissue of mice.

The purpose of the present study was to test Enterogenin for genotoxicity. The micronuclearic test of Schmid W. was used. Enterogenin was introduced intraperitoneally, once and/or twice in two doses on male and female mice of the BDF 1 hybrid line. The positive controls were injected with clastogenic agent. Bone marrow smear preparations were made, determining the ratio between the normochromic erythrocytes (NCE) and polychromic erythrocytes (PCE), and the number of micronuclearic erythrocytes (MNE). Enterogenin has no genotoxic effect on the hemopoietic tissue of bone marrow of mice the ratio NCE/PCE being within the reference values. The preparation showed no clastogenic effect (p > 0.05), compared to the controls, as the number of PCE with micronuclei did not change 24, 48 and 72 hours after treatment with the two studied doses.

[Diagnostic effect of bronchoalveolar lavage in early lung injury caused by enterogenic infection in rat].

OBJECTIVE: To study the diagnostic effect of bronchoalveolar lavage in early lung injury by observing changes in inflammatory mediators in early lung injury caused by enterogenic infection. METHODS: Eighty-four Sprague-Dawley rats were randomly divided into infection group and sham-operation group. Cecal ligation and perforation was utilized to produce abdominal infection in rats. Six groups were sacrificed respectively at 0, 24, 48, 72, 96, 120 hours after operation. The differential cell count in bronchoalveolar lavage fluid (BALF) was assessed. The concentrations of endotoxin, phospholipase A2 (PLA2) and tumor necrosis factor-alpha (TNF-alpha) in BALF, lung and plasma were assayed. RESULTS: The neutrophil percentage of BALF increased progressively. The concentrations of endotoxin, PLA2 and TNF-alpha in BALF, lung and plasma were significantly increased. The levels of endotoxin and PLA2 in lung tissue were respectively correlated positively with those in BALF and plasma (BALF and lung: r=0.904, P<0.05; BALF and plasma: r=0.895, P<0.05; lung and plasma: r=0.946, P<0.01). Significant positive correlation was also present between the TNF-alpha levels in BALF and lung (r=0.952 P<0.01), but not between the TNF-alpha level in plasma and that in lung or BALF (r=0.684, r=0.608, both P>0.05). CONCLUSION: The examinations of bronchoalveolar lavage may help discover early lung injury caused by enterogenic infection.

Antiradiation mixture and ionic-bond product of AET and adenyl nucleotides.

These experiments continued our line of inquiry into low-toxicity high-effectiveness radiomodifying agents obtained by binding radioprotective substances to biogenic and biologically active components or antidotes. Testes for toxicity and radioprotective effects were three preparations combining within one molecule AET and an adenyl nucleotide (AMP, ADP, or ATP). Ionic-bond formation was shown to have advantages over concomitant administration of AET and adenosine phosphoric acids as mixtures. The evidence obtained supports the rational foundation of the concept being developed by our laboratory.