Effects Of Nucleotide Analogues Research Articles

Poor clinical and virological outcome of nucleos(t)ide analogue monotherapy in HBV/HDV co-infected patients.

Co-infection of Hepatitis B (HBV) and Delta viruses (HDV) represent the most severe form of viral hepatitis. While treatment with pegylated Interferon alpha (PEG-IFNα) is well established, therapy with nucleoside or nucleotide analogues (NA) has been a matter of debate. We aimed to investigate the role of NA treatment in a well-defined single centre cohort.In a retrospective approach, we observed 53 HDV RNA positive and/or anti-HDV-positive patients recruited at a German referral centre between 2000 and 2019. Patients were followed for at least 3 months (mean time of follow up: 4.6 years; range: 0.2–14.1 years). Patients who had liver transplantation or hepatocellular carcinoma at the time of presentation were excluded. 43% (n = 23) were treated with NA, 43% (n = 23) received IFNα-based therapies and 13% (n = 7) were untreated.Liver cirrhosis was already present in 53% (28/53) of patients at first presentation. During follow-up, liver-related endpoints developed in 44% of all patients (n = 23). NA-treatment was associated with a significantly worse clinical outcome (P = .01; odds ratio [OR] = 4.92; CI = 1.51–16.01) compared to both, untreated (P = .38; OR = 0.46; CI = 0.80–2.61) and IFNα-based-treated patients (P = .04; OR = 0.29; CI = 0.89–0.94) in univariate logistic regression analysis. HBsAg levels declined by more than 50% during NA-based therapy in only 7 cases (7/23; mean time: 3.6 years; range: 0.8–8.5 years) and during IFNα-based therapy in 14 cases (14/23; mean time: 2.8 years, range 0.7–8.5 years). HDV RNA became undetectable during follow up in 30% of patients receiving NA alone (7/23; mean time: 5.0 years; range: 0.6–13.5 years), in 35% of patients receiving IFNα-based therapy (8/23; mean time: 2.9 years, range: 0.3–7.6 years).The effect of NA in patients with HBV/HDV co-infection is limited. Treatment with NA was associated with a higher likelihood of clinical disease progression. Interferon alpha therapy was beneficial in reducing liver complications and improves long-term outcome.

Determination of Microtubule Lattice Parameters from Cryo-electron Microscope Images Using TubuleJ.

The α-β tubulin heterodimer undergoes subtle conformational changes during microtubule assembly. These can be modulated by external factors, whose effects on microtubule structure can be characterized on 2D views obtained by cryo-electron microscopy. Analysis of microtubule images is facilitated if they are straight enough to interpret and filter their image Fourier transform, which provide useful information concerning the arrangement of tubulin molecules inside the microtubule lattice. Here, we describe the use of the TubuleJ software to straighten microtubules and determine their lattice parameters. Basic 3D reconstructions can be performed to evaluate the relevance of these parameters. This approach can be used to analyze the effects of nucleotide analogues, drugs or MAPs on microtubule structure, or to select microtubule images prior to high-resolution 3D reconstructions.

Chinese herbal medicine combined with nucleotide analogues for compensated HBV-related cirrhosis: a systematic review of randomized controlled trials

Abstract Objective To systematically evaluate the effectiveness and safety of Chinese herbal medicine (CHM) plus nucleotide analogues (NAs) for treating compensated HBV-related cirrhosis, the early stage of cirrhosis. Methods PubMed, Cochrane library, China Network Knowledge Infrastructure Database (CNKI), Chinese Scientific Journals Database (VIP), Wan Fang Database and Sino-Med Database were searched. Randomized controlled trials (RCTs) and quasi-RCTs comparing NAs and NAs plus CHM therapy on patients with compensated HBV-related cirrhosis were included. Two reviewers independently extracted information and assessed the methodological quality of the trials. Different CHM herbal formulas used in the trials were considered. Primary meta-analysis was conducted when there were at least two trials comparing the same CHM formula. Results Forty-five trials comprising 3497 participants were included. The quality of most of the trials was moderate or low. Twenty-six herbal formulations were identified. A meta-analysis was conducted for compound Biejia Ruangan (FFBJ), Dahuang Zhechong (DHZC), and Fuzheng Huayu (FZHY). The results of the subgroup analysis showed a beneficial effect of FFBJ plus entecavir (ETV), and DHZC plus adefovir dipivoxil (ADV) on hyaluronic acid (HA); FFBJ plus ADV on laminin (LN); and FZHY plus ADV on HA, LN, and precollagen type III (PC-III). The results from other studies suggested significant benefits of CHM plus NAs compared with NAs alone, except those on albumin (ALB). None of the trials evaluated the quality of life or reported severe adverse events. Conclusions A positive effect was found for FFBJ plus ETV, DHZC plus ADV, and FZHY plus ADV on HA; FZHY plus ADV and FFBJ plus ADV on LN; and FZHY plus ADV on PCIII compared with the effects of NAs used alone.

Hepatitis B virus mutation may play a role in hepatocellular carcinoma recurrence: A systematic review and meta-regression analysis.

A number of studies have confirmed that antiviral therapy with nucleotide analogs (NAs) can improve the prognosis of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after curative therapy. However, what factors affected the prognosis of HBV-HCC after removal of the primary tumor and inhibition of HBV replication? A meta-regression analysis was conducted to explore the prognostic factor for this subgroup of patients. MEDLINE, EMBASE, Web of Science, and Cochrane library were searched from January 1995 to February 2014 for clinical trials evaluating the effect of NAs on the prognosis of HBV-HCC after curative therapy. Data were extracted for host, viral, and intervention information. Single-arm meta-analysis was performed to assess overall survival (OS) rates and HCC recurrence. Meta-regression analysis was carried out to explore risk factors for 1-year OS rate and HCC recurrence for HBV-HCC patients after curative therapy and antiviral therapy. Fourteen observational studies with 1284 patients met the inclusion criteria. Influential factors for prognosis of HCC were mainly baseline HBeAg positivity, cirrhotic stage, advanced Tumor-Node-Metastasis (TNM) stage, macrovascular invasion, and antiviral agent type. The 1-year OS rate decreased by more than four times (coefficient -4.45, P<0.001) and the 1-year HCC recurrence increased by more than one time (coefficient 1.20, P=0.003) when lamivudine was chosen for HCC after curative therapy, relative to entecavir for HCC. HBV mutation may play a role in HCC recurrence. Entecavir or tenofovir, a high genetic barrier to resistance, should be recommended for HBV-HCC patients.

Chinese herbal medicine combined with nucleotide analogues for compensated HBV-related cirrhosis: a systematic review of randomized controlled trials

ObjectiveTo systematically evaluate the effectiveness and safety of Chinese herbal medicine (CHM) plus nucleotide analogues (NAs) for treating compensated HBV-related cirrhosis, the early stage of cirrhosis. MethodsPubMed, Cochrane library, China Network Knowledge Infrastructure Database (CNKI), Chinese Scientific Journals Database (VIP), Wan Fang Database and Sino-Med Database were searched. Randomized controlled trials (RCTs) and quasi-RCTs comparing NAs and NAs plus CHM therapy on patients with compensated HBV-related cirrhosis were included. Two reviewers independently extracted information and assessed the methodological quality of the trials. Different CHM herbal formulas used in the trials were considered. Primary meta-analysis was conducted when there were at least two trials comparing the same CHM formula. ResultsForty-five trials comprising 3497 participants were included. The quality of most of the trials was moderate or low. Twenty-six herbal formulations were identified. A meta-analysis was conducted for compound Biejia Ruangan (FFBJ), Dahuang Zhechong (DHZC), and Fuzheng Huayu (FZHY). The results of the subgroup analysis showed a beneficial effect of FFBJ plus entecavir (ETV), and DHZC plus adefovir dipivoxil (ADV) on hyaluronic acid (HA); FFBJ plus ADV on laminin (LN); and FZHY plus ADV on HA, LN, and precollagen type III (PC-III). The results from other studies suggested significant benefits of CHM plus NAs compared with NAs alone, except those on albumin (ALB). None of the trials evaluated the quality of life or reported severe adverse events. ConclusionsA positive effect was found for FFBJ plus ETV, DHZC plus ADV, and FZHY plus ADV on HA; FZHY plus ADV and FFBJ plus ADV on LN; and FZHY plus ADV on PCIII compared with the effects of NAs used alone.

Chemical modification reveals involvement of different sites for nucleotide analogues in the phosphatase activity of the red cell calcium pump.

The calcium pump of plasma membranes catalyzes the hydrolysis of ATP and phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter activity requires the presence of ATP and/or calmodulin, and Ca2+ [22, 25]. We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca2+-pNPPase activity. Treatment with fluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dependent pNPPase activity. The nucleotide analogue eosin-Y had an inhibitory effect on calmodulin-dependent pNPPase (Kieosin-Y = 2 microM). FITC treatment increased Kieosin-Y 15 times. Acetylation of lysine residues with N-hydroxysuccinimidyl acetate inactivates Ca2+-ATPase by modifying the catalytic site, and impairs stimulation by modulators by modifying residues outside this site [9]. Acetylation suppressed the ATP-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetylation cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase. The decreased affinity for eosin-Y of the FITC-modified enzyme, suggests that the sites for these two molecules are closely related but not overlapped. Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATP-pNPPase. Since calmodulin binds to E1, the E1 conformation or the E2 if E1 transition would be involved during calmodulin-dependent pNPPase activity.

Characterization of the recombinant human prostanoid DP receptor and identification of L‐644,698, a novel selective DP agonist

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D2 (PGD2) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway. 2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (Ki) of 0.9 nM. This Ki value was comparable to the Ki values obtained in this study for ligands that have previously shown high affinity for DP: PGD2 (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM). 3. L-644,698 was found to be a full agonist with an EC50 value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC50): PGD2 (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM). 4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP1 (> 25,400 fold), EP2 (approximately 300 fold), EP3-III (approximately 4100 fold), EP4 (approximately 10000 fold), FP (> 25,400 fold), IP (> 25,400 fold) and TP (> 25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described. 5. PGJ2 and delta12-PGJ2, two endogenous metabolites of PGD2, were also tested in this system and shown to be effective agonists with Ki and EC50 values in the nanomolar range for both compounds. In particular, PGJ2 was equipotent to known DP specific agonists with a Ki value of 0.9 nM and an EC50 value of 1.2 nM.

Methenyltetrahydrofolate Cyclohydrolase Catalyzes the Synthesis of (6S)-5-Formyltetrahydrofolate

The methenyltetrahydrofolate cyclohydrolase activity of the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain catalyzes the conversion of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate. We have observed that in the presence of this domain, 10-formyltetrahydrofolate is converted to 5-formyltetrahydrofolate. The effects of nucleotide analogs on the initial rates of this reaction were found to be similar to their effects on the cyclohydrolase activity, establishing that 5-formyltetrahydrofolate production is dependent on the cyclohydrolase. The specific activity of 5-formyltetrahydrofolate production is approximately 7 × 104-fold lower than that of the cyclohydrolase activity but can be used to obtain quantitative conversion of 10-formylH4folate to 5-formyltetrahydrofolate, in mg amounts. This “side-reaction” may contribute to thein vivoproduction of (6S)-5-formyltetrahydrofolate.

Effects of nucleotide analogs on ATP hydrolysis by P‐type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+‐ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P‐type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na++ K+) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H+‐ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7‐chloro‐4‐nitrobenz‐2‐oxa‐1.3‐diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′‐p‐fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na++ K+) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′‐o‐(4‐benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′‐o‐(4‐benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na++ K+) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′‐adenylylimidodiphosphate for use as an ATP binding probe.

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na+ + K+) ATPase and maize root H+-ATPase

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na+ + K+) ATPase and maize root H+-ATPase

Effect of nucleotide analogues on rotavirus transcription and replication

The effects of several nucleoside and nucleoside triphosphate analogues were studied on rotavirus replication and transcription. Nucleoside triphosphate analogues modified at sugar residues were capable of inhibiting in vitro transcription, including adenosine-9-β- d-arabinofuranoside 5′-triphosphate, 3′-deoxyadenosine 5′-triphosphate, adenosine 5′-triphosphate 2′,3′-dialdehyde, guanosine 5′-triphosphate 2′,3′-dialdehyde, and cytosine-9-β- d-arabinofuranoside 5′-triphosphate. Two dialdehyde derivatives, adenosine 5′-triphosphate 2′,3′ dialdehyde and guanosine 5′-triphosphate 2′,3′-dialdehyde, were irreversible inhibitors, forming a stable complex with the viral polypeptide VP3. The effect of the corresponding nucleosides of the inhibitory analogues was studied in SA-11 rotavirus-infected MA-104 cells. Adenosine-9-β- d-arabinofuranoside and 3′-deoxyadenosine were effective inhibitors of RNA synthesis, an effect that could be due to their inhibition of viral transcription.

The effect of nucleotide analogs on cell-free gene expression.

The effects of the following pyrimidine nucleoside 5'-triphosphates: f5 UTP, br5 UTP, rTTP, s2 UTP, s4 UTP and s2 CTP on cell-free expression of the beta-galactosidase gene in lambda h80dlac DNA as well as the galactokinase gene in plasmid 01-14 were investigated. Only rTTP could substitute UTP in cell-free gene expression without restriction. Combinations of the other analogs with their respective natural congeners led to inhibition of gene expression. All analogs were found to inhibit transcription. Whereas br5 UTP and s4 UTP did not affect translation, mRNA containing s2 UMP or s2 CMP residues respectively was found to function poorly in translation. Only in the case of f5 UTP could ambiguitive behaviour be demonstrated. Whether mispairing of f5 UMP residues, responsible for this ambiguity takes place in transcription or in translation, could not be decided.

Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI.

The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.

Ouabain binding to ( [formula omitted] effects of nucleotide analogues and ethacrynic acid

To determine the mechanism of Na +, M 2+ and ATP stimulated [ 3H]ouabain binding to ( Na + + K +)- ATPase (ATP phosphohydrolase, EC 3.6.1.3) the effects on this reaction of ethacrynic acid and analogues of ATP were investigated. β,7-Methylene-ATP inhibited [ 3H]ouabain binding to this enzyme under all conditions tested. This inhibition was synergistic with that of Na +, and Na + acted to increase the apparent affinity of the enzyme for β,γ-methylene-ATP. β,γ-Methylene ADP also inhibited [ +H]ouabain binding to this enzyme. Similarly, the overall effect of β,γ-imido-ATP on [ 3H]ouabain binding to guinea pig kidney ( Na − − K +)- ATPase was inhibitory. Ethacrynic acid prolonged transient pulses of [ 3H]ouabain binding supported by ATP or ADP. Ethacynic acid produced this effect by stabilizing the form of the enzyme which interacted with ouabain rather than by affecting the rates at which ouabain was bound to or dissociated from the enzyme. The data suggest that nucleotides with a stable terminal phosphate bond cannot support [ 3H]ouabain binding. These observations are consistent with the hypothesis that Na +-stimulated phosphorylation of the (Na + + K +)- ATPase is a primary event in the Na +-stimulated binding of [ 3H]-ouabain to this enzyme.

DNA synthesis in nucleotide-permeable Escherichia coli cells. The effects of nucleotide analogues on DNA synthesis.

DNA synthesis in nucleotide-permeable Escherichia coli cells. The effects of nucleotide analogues on DNA synthesis.