scholarly journals Site-Specific Cleavage of RNAs Derived from the PIM1 3′-UTR by a Metal-Free Artificial Ribonuclease

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 807 ◽  
Author(s):  
Felix Zellmann ◽  
Laura Thomas ◽  
Ute Scheffer ◽  
Roland Hartmann ◽  
Michael Göbel
Keyword(s):  
Competitive Inhibition ◽  
Reaction Rates ◽  
Phosphate Esters ◽  
Rna Cleavage ◽  
Site Specificity ◽  
Cleavage Rate ◽  
Short Oligonucleotide ◽  
A Cell ◽  
Artificial Nuclease ◽  
Oligonucleotide Conjugates

Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.

scholarly journals Protein/oligonucleotide conjugates as a cell specific PNA carrier

2001 ◽  
Vol 1 (1) ◽  
pp. 217-218 ◽  
Author(s):  
K. Obara ◽  
T. Ishihara ◽  
T. Akaike ◽  
A. Maruyama
Keyword(s):  
A Cell ◽  
Oligonucleotide Conjugates

Absence of glucokinase in Methanomonas sp. as a cause for their inability to grow on glucose

1972 ◽  
Vol 18 (12) ◽  
pp. 1907-1913 ◽  
Author(s):  
Kei Amemiya
Keyword(s):  
Phosphate Esters ◽  
Resting Cells ◽  
Obligate Methylotroph ◽  
Enzymatic Assays ◽  
Autotrophic Bacteria ◽  
Toxic Product ◽  
A Cell ◽  
Flow Through ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Autotrophic Bacterium

Many obligate autotrophic bacteria can be grown on glucose using a dialysis flow-through system. Methanomonas methanooxidans, an obligate methylotroph, exhibits many of the properties of an obligate autotrophic bacterium but we have been unable to grow it on glucose using dialysis. In the obligate autotrophic bacteria, the dialysis procedure seems to be removing a toxic product of glucose metabolism but this does not seem to be the case with the methylotroph. Enzymatic assays on a cell-free extract from methane-grown or methane plus glucose-grown cells showed only phosphoglucoisomerase activity, while glucokinase and glucose-6-phosphate dehydrogenase activity were not detected. Studies with resting cells showed that glucose was not oxidized, although the phosphate esters of glucose, fructose, ribose, and gluconate were oxidized. CO2 fixation occurred only in the presence of glucose-6-phosphate. The rate of oxygen consumed and CO2 fixed on glucose-6-phosphate were almost identical with that when methanol was used as the substrate. When the phosphate esters of glucose, fructose, and ribose were used as the sole energy source, only glucose-6-phosphate supported growth to any extent; in fact, the amount of growth was essentially the same as that obtained with methanol. The results from this study suggest that the inability of this organism to grow on glucose may be due to the absence of adequate glucokinase.

scholarly journals Salivary apyrase of Rhodnius prolixus. Kinetics and purification

1986 ◽  
Vol 233 (3) ◽  
pp. 885-891 ◽  
Author(s):  
J J F Sarkis ◽  
J A Guimarães ◽  
J M C Ribeiro
Keyword(s):  
Atp Hydrolysis ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Rhodnius Prolixus ◽  
Exchange Chromatography ◽  
Phosphate Esters ◽  
Inorganic Pyrophosphatase ◽  
Blood Sucking ◽  
Specific Activities

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5′-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.

Novel Short Oligonucleotide Conjugates as Inhibitors of Human Telomerase

ChemInform ◽  
2004 ◽  
Vol 35 (9) ◽  
Author(s):  
Krisztina Pongracz ◽  
Shihong Li ◽  
Brittney-Shea Herbert ◽  
Ronald Pruzan ◽  
Ellen Wunder ◽  
...  
Keyword(s):  
Short Oligonucleotide ◽  
Human Telomerase ◽  
Oligonucleotide Conjugates

Pore-Spanning PPy(DBS) as a Voltage-Gated Synthetic Membrane Ion Channel

2016 ◽  
Author(s):  
Travis M. Hery ◽  
Vishnu-Baba Sundaresan
Keyword(s):  
Ion Transport ◽  
Reaction Rates ◽  
Transport Rate ◽  
Electrical Field ◽  
A Cell ◽  
The Given ◽  
First Time ◽  
Protein Reaction ◽  
Applied Electrical Field ◽  
Reduced State

The transport of monovalent cations across a suspended PPy(DBS) polymer membrane in an aqueous solution as a function of its redox state is investigated. Maximum ion transport is found to occur when PPy(DBS) is in the reduced state, and minimum transport in the oxidized state. No deviation in the dynamics of ion transport based on the direction of the applied electrical field is observed. Additionally, it is found that ion transport rates linearly increased proportional to the state of reduction until a steady state is reached when the polymer is fully reduced. Therefore controlled, bidirectional ion transport is for the first time demonstrated. The effect of aqueous Li+ concentration on ion transport in the fully reduced state of the polymer is studied. It is found that ion transport concentration dependence follows Michaelis-Menten kinetics (which models protein reaction rates, such as those forming ion channels in a cell membrane) with an r2 value of 0.99. For the given PPy(DBS) polymer charge density and applied potential across the membrane, the maximum possible ion transport rate per channel is found to be 738 ions per second and the Michaelis constant, representing the concentration at which half the maximum ion transport rate occurs, is 619.5mM.

scholarly journals Evaluation of 3-Chlorobenzoate 1,2-Dioxygenase Inhibition by 2- and 4-Chlorobenzoate with a Cell-Based Technique

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 106 ◽  
Author(s):  
Emelyanova ◽  
Solyanikova
Keyword(s):  
Competitive Inhibition ◽  
Enzymatic Reaction ◽  
Oxygen Electrode ◽  
Electrochemical Reactor ◽  
Rhodococcus Opacus ◽  
Microbial Sensor ◽  
Rhodococcus Opacus 1Cp ◽  
Excess Substrate ◽  
A Cell ◽  
The Hill

The electrochemical reactor microbial sensor with the Clark oxygen electrode as the transducer was used for investigation of the competition between 3-chlorobenzoate (3-CBA) and its analogues, 2- and 4-chlorobenzoate (2-CBA and 4-CBA), for 3-chlorobenzoate-1,2-dioxygenase (3-CBDO) of Rhodococcus opacus 1CP cells. The change in respiration of freshly harvested R. opacus 1CP cells in response to 3-CBA served as an indicator of 3-CBDO activity. The results obtained confirmed inducibility of 3-CBDO. Sigmoidal dependency of the rate of the enzymatic reaction on the concentration of 3-CBA was obtained and positive kinetic cooperativity by a substrate was shown for 3-CBDO. The Hill concentration constant, S0.5, and the constant of catalytic activity, Vmax, were determined. Inhibition of the rate of enzymatic reaction by excess substrate, 3-CBA, was observed. Associative (competitive inhibition according to classic classification) and transient types of the 3-CBA-1,2-DO inhibition by 2-CBA and 4-CBA, respectively, were found. The kinetic parameters such as S0.5i and Vmaxi were also estimated for 2-CBA and 4-CBA. The disappearance of the S-shape of the curve of the V versus S dependence for 3-CBDO in the presence of 4-CBA was assumed to imply that 4-chlorobenzoate had no capability to be catalytically transformed by 3-chlorobenzoate-1,2-dioxygenase of Rhodococcus opacus 1CP cells.

scholarly journals Biosynthesis of heparin. Modulation of polysaccharide chain length in a cell-free system

1988 ◽  
Vol 254 (2) ◽  
pp. 571-578 ◽  
Author(s):  
K Lidholt ◽  
J Riesenfeld ◽  
K G Jacobsson ◽  
D S Feingold ◽  
U Lindahl
Keyword(s):  
Competitive Inhibition ◽  
Alkali Treatment ◽  
Polymerization Reaction ◽  
Chain Elongation ◽  
Free System ◽  
Polysaccharide Chain ◽  
Predominant Component ◽  
A Cell ◽  
Final Length ◽  
High Concentrations

The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.

Novel Short Oligonucleotide Conjugates as Inhibitors of Human Telomerase

2003 ◽  
Vol 22 (5-8) ◽  
pp. 1627-1629 ◽  
Author(s):  
Krisztina Pongracz ◽  
Shihong Li ◽  
Brittney-Shea Herbert ◽  
Ronald Pruzan ◽  
Ellen Wunder ◽  
...  
Keyword(s):  
Short Oligonucleotide ◽  
Human Telomerase ◽  
Oligonucleotide Conjugates

scholarly journals Metabolism of an Alkyl Polyamine Analog by a Polyamine Oxidase from the Microsporidian Encephalitozoon cuniculi

2009 ◽  
Vol 53 (6) ◽  
pp. 2599-2604 ◽  
Author(s):  
Cyrus J. Bacchi ◽  
Nigel Yarlett ◽  
Evangeline Faciane ◽  
Xiangdong Bi ◽  
Donna Rattendi ◽  
...  
Keyword(s):  
Oxidase Activity ◽  
Competitive Inhibition ◽  
Amine Oxidase ◽  
Reaction Rates ◽  
Competitive Inhibitor ◽  
Host Cells ◽  
Nuclear Magnetic Resonance Analysis ◽  
Encephalitozoon Cuniculi ◽  
Resonance Analysis ◽  
Polyamine Analog

ABSTRACT Encephalitozoon cuniculi is a microsporidium responsible for systemic illness in mammals. In the course of developing leads to new therapy for microsporidiosis, we found that a bis(phenylbenzyl)3-7-3 analog of spermine, 1,15-bis{N-[o-(phenyl)benzylamino}-4,12-diazapentadecane (BW-1), was a substrate for an E. cuniculi amine oxidase activity. The primary natural substrate for this oxidase activity was N′-acetylspermine, but BW-1 had activity comparable to that of the substrate. As the sole substrate, BW-1 gave linear reaction rates over 15 min and K m of 2 μM. In the presence of N′-acetylspermine, BW-1 acted as a competitive inhibitor of oxidase activity and may be a subversive substrate, resulting in increased peroxide production. By use of 13C-labeled BW-1 as a substrate and nuclear magnetic resonance analysis, two products were determined to be oxidative metabolites, a hydrated aldehyde or dicarboxylate and 2(phenyl)benzylamine. These products were detected after exposure of 13C-labeled BW-1 to E. cuniculi preemergent spore preparations and to uninfected host cells. In previous studies, BW-1 was curative in a rodent model of infection with E. cuniculi. The results in this study demonstrate competitive inhibition of oxidase activity by BW-1 and support further studies of this oxidase activity by the parasite and host.

Citrate transport in proximal cell line

1992 ◽  
Vol 263 (1) ◽  
pp. C220-C225 ◽  
Author(s):  
D. Law ◽  
K. S. Hering-Smith ◽  
L. L. Hamm
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Transport Process ◽  
Competitive Inhibition ◽  
Basolateral Membrane ◽  
Opossum Kidney ◽  
Cell Monolayers ◽  
Citrate Transport ◽  
Sodium Dependent ◽  
A Cell

Citrate uptake into kidney proximal tubules occurs via an apical dicarboxylate transporter and a poorly characterized process in the basolateral membrane. We used OK cells, a cell line derived from opossum kidney, to study citrate transport in proximal tubule-like cells. Citrate uptake into cell monolayers was studied using [14C]citrate with [3H]mannitol as a volume marker. Citrate uptake into these cells was sodium dependent and saturable with increasing concentrations of citrate. In contrast to previous models, citrate transport was altered minimally by changes in pH from 6.2 to 7.0 and increased at pH 7.4 to 7.8. A variety of di- and tricarboxylates were tested for interaction with citrate transport. The dicarboxylates succinate, malate, and oxaloacetate at 1 mM concentration inhibited citrate uptake minimally (uptake at least 80% of control); one dicarboxylate, alpha-ketoglutarate, did inhibit citrate uptake significantly. In contrast, the tricarboxylates isocitrate and tricarballylate inhibited citrate uptake significantly, indicating probable competitive inhibition with the transport process. These characteristics are distinctly different from those of the apical membrane dicarboxylate transporter. 1,2,3-Benzenetricarboxylic acid, an inhibitor of the mitochondrial tricarboxylate transporter, did not alter citrate uptake. In conclusion, the OK proximal cell line exhibits a novel citrate transport process compared with the apical transport of citrate described in most proximal systems. This transport process probably involves the trivalent species of citrate in contrast to the usual predominant transport of divalent citrate. This transport process may represent a process similar to that in the basolateral membrane of the proximal tubule.

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