Koumine modulates spinal microglial M1 polarization and the inflammatory response through the Notch-RBP-Jκ signaling pathway,ameliorating diabetic neuropathic pain in rats

BackgroundDiabetic neuropathic pain (DNP), a complication of diabetes, has serious impacts on human health. As the pathogenesis of DNP is very complex, clinical treatments for DNP is limited. Koumine (KM) is an active ingredient extracted from Gelsemium elegans Benth. that exerts an inhibitory effect on neuropathic pain (NP) in several animal models.PurposeTo clarify the anti-NP effect of KM on rats with DNP and the molecular mechanisms involving the Notch- Jκ recombination signal binding protein (RBP-Jκ) signaling pathway.MethodsMale Sprague-Dawley rats were administered streptozocin (STZ) by intraperitoneal injection to induce DNP. The effect of KM on mechanical hyperalgesia in rats with DNP was evaluated using the Von Frey test. Microglial polarization in the spinal cord was examined using western blotting and quantitative real-time PCR. The Notch-RBP-Jκ signaling pathway was analysed using western blotting.ResultsKM attenuated DNP during the observation period. In addition, KM alleviated M1 microglial polarization in STZ-induced rats. Subsequent experiments revealed that Notch-RBP-Jκ signaling pathway was activated in the spinal cord of rats with DNP, and the activation of this pathways was decreased by KM. Additionally, KM-mediated analgesia and deactivation of the Notch-RBP-Jκ signaling pathway were inhibited by the Notch signaling agonist jagged 1, indicating that the anti-DNP effect of KM may be regulated by the Notch-RBP-Jκ signaling pathway.ConclusionsKM is a potentially desirable candidate treatment for DNP that may inhibit microglial M1 polarization through the Notch-RBP-Jκ signaling pathway.     

Kinetic and structural studies on the interactions of Torpedo californica acetylcholinesterase with two donepezil-like rigid analogues

Acetylcholinesterase inhibitors were introduced for the symptomatic treatment of Alzheimer’s disease (AD). Among the currently approved inhibitors, donepezil (DNP) is one of the most preferred choices in AD therapy. The X-ray crystal structures of Torpedo californica AChE in complex with two novel rigid DNP-like analogs, compounds 1 and 2, have been determined. Kinetic studies indicated that compounds 1 and 2 show a mixed-type inhibition against TcAChE, with K_i values of 11.12?±?2.88 and 29.86?±?1.12?nM, respectively. The DNP rigidification results in a likely entropy-enthalpy compensation with solvation effects contributing primarily to AChE binding affinity. Molecular docking evidenced the molecular basis for the binding of compounds 1 and 2 to the active site of β-secretase-1. Overall, these simplified DNP derivatives may represent new structural templates for the design of lead compounds for a more effective therapeutic strategy against AD by foreseeing a dual AChE and BACE-1 inhibitory activity.     

Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides.     

Elucidation of the Hydrolytical Properties of α-Hydroxybenzylphosphonates as a New Potential Pro-Oligonucleotide Concept

Abstract

The synthesis of Fpmp-protected α-hydroxybenzylphosphonate modified oligonucleotides as potential new pro-oligonucleotides is described. The proposed hydrolytic pathways of the oligonucleotides were studied using two dimers 2 and 4 and the tetramer 6 containing one α-hydroxybenzyl modification as model compounds.     

Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

Abstract

The effect of 2, 4-dinitrophenol (DNP) on the extracelluar polysaccharides (EPS), cell surface charge, and the hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene was evaluated. DNP treatment did not influence cell surface charge and EPS production, but had a significant effect on hydrophobicity of both hydrophilic (p = 0.05) and hydrophobic (p = 0.01) cultures. Significant reduction in the attachment of all the six cultures to glass (p = 0.02) and polystyrene (p = 0.03) was observed after DNP treatment. Moreover, hydrophobicity but not the cell surface charge or EPS production influenced bacterial cell attachment to glass and polystyrene. From this study, it was evident that DNP treatment influenced bacterial cell surface hydrophobicity, which in turn, reduced bacterial adhesion to surfaces.     

Sul Controllo Della Glicolisi Nel Lievito

Abstract

On the control of carbohydrate utilization in yeast. — The results of a previous investigation showed that in higher plants the stimulating action of 2,4 dinitrophenol (DNP) on oxygen uptake and glycolysis is accompained by a fall of the level of reducing sugars, due to an increase of their respiratory utilization, and thus — according to every evidence — of the rate of hexose phosphate synthesis.

In the present work, the occurrence of a similar phenomenon in yeast (where the inhibiting effect of DNP on glucose uptake is not so much marked as in higher plant tissue) was investigated.

Here again DNP, at a 10^-4M concentration, induced a rapid decrease of the disaccaride trehalose and of glycogen, such as to account for the increased rate of respiration and of fermentation. The ratio between the contributions to CO₂ of Carbons 1 and respectively 6 of glucose was not significantly changed by DNP, which suggests that at least part of the DNP induced increase of glycolysis was mediated by the Embden Meyerhof pathway, and thus that a larger amount of fructose diphosphate was formed in the presence of the uncoupler.

In other experiments the effects of DNP on the dissimilation of C¹⁴ labeled glucose, glycerol and pyruvate to CO₂ and ethanol, and on the incorporation of the radioactive isotope into various fractions, 15 minutes after feeding the labeled substrates, was investigated. It was found that:

1) Glucose and glycerol uptake is not markedly inhibited by DNP at the concentration employed (^10–4M).

2) In the absence of DNP, a considerable portion of the radioactivity fed as glucose or glycerol and taken up by the yeast cells is recovered in the glycogen and trehalose fractions. (35% of the glucose, and 22% of the glycerol taken up). This is also observed for carbons 2 and 3, but not for carbon 1 of pyruvate. This indicates a reversibility of the glycolitic processes comprehended in the region between phospho-enol pyruvate andpolysac-carides; while the pyruvate kinase reaction appears to represent a sharp barrier at the « lower » end of glycolysis.

3) DNP almost completely inhibited the incorporation of C¹⁴ from glucose and glycerol into glycogen and trehalose, although it increased the rate of its dissimilation to CO₂ and ethanol. The total amount of glucose and glycerol transformed in the various metabolites (and thus — according to every evidence — phosphorylated) was somewhat lowered and proteins synthesis severely depressed. These effects are interpreted as due to the uncoupling action of DNP at the mitochondrial level, and to the consequent general decrease of the ATP and UTP levels required for protein and for polysaccharide synthesis.     

Synthesis and Enzymatic Studies of Modified Oligonucleotides with Abiological Monomer Units

Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block¹ and the modified PNA-spacer (acyclic serinol nucleoside)² can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.     

Quantitative One Step Derivatization of Oligonucleotides by a Fluorescent Label Through Abasic Site Formation

Abstract

Reaction of abasic site-containing oligonucleotides with an oxyamino fluorescent label is described. The reaction represents an efficient method to functionalize oligonucleotides at preselected positions.     

Comparison of Physicochemical and Biological Properties of (o-Carboran-1-YL)methylphosphonate and Methylphosphonate Oligonucleotides

Abstract

(o-Carboran-1-yl)methylphosphonate (CBMP) oligonucleotides demonstrate advantages in their physicochemical and biological characteristics over the corresponding methylphosphonate oligonucleotides.     

Synthesis and Properties of Some (2′-5′) Linked Dinucleoside Monophosphates Modified with 3′-Difluoromethylene Groups

Abstract

The title dimers were prepared to investigate conditions required for the synthesis of 3′-difluoromethylene modified oligonucleotides on solid support. As a result a new synthetic cycle was developed that enabled the solid phase synthesis of the modified oligonucleotides.     

Preparation of Oligonucleotides Containing Non-natural Base Analogues

Abstract

The preparation of a protected derivative of 2-aza- 2′-deoxyinosine carrying a photolabile protecting group is described. The new derivative is useful to prepare oligonucleotides containing 2-azahypoxanthine. The synthesis of oligonucleotides containing 2-fluorohypoxanthine and O⁴-alkylthymine is also described.     

Boron nitride nanotubes for gene silencing

BackgroundNon-viral gene delivery is increasingly investigated as an alternative to viral vectors due to low toxicity and immunogenicity, easy preparation, tissue specificity, and ability to transfer larger sizes of genes.MethodsIn this study, boron nitride nanotubes (BNNTs) are functionalized with oligonucleotides (oligo-BNNTs). The morpholinos complementary to the oligonucleotides attached to the BNNTs (morpholino/oligo-BNNTs) are hybridized to silence the luciferase gene. The morpholino/oligo-BNNTs conjugates are administered to luciferase-expressing cells (MDA-MB-231-luc2) and the luciferase activity is monitored.ResultsThe luciferase activity is decreased when MDA-MB-231-luc2 cells were treated with morpholino/oligo-BNNTs.ConclusionsThe study suggests that BNNTs can be used as a potential vector to transfect cells.General significanceBNNTs are potential new nanocarriers for gene delivery applications.     

Synthesis and Biophysical Studies of Modified Oligonucleotides Containing Acyclic Amino Alcohol Nucleoside Analogs

Abstract

Novel serine derivative of thymine was prepared and incorporated into oligonucleotides. These modified oligonucleotides were studied for their binding affinity with complementary DNA/RNA.     

Covalent Attachment of Methylene Blue to Oligonucleotides

Abstract

The synthesis and application of oligonucleotides derivatized by methylene blue are described. For that, a carboxylated methylene blue derivative was synthesized and transformed into an activated N-hydroxysuccinimidoester. The activated ester was reacted with 5′-aminoalkylated oligonucleotides. The labelled oligonucleotides were isolated and characterized both by reversed phase HPLC and MALDI mass spectrometry. Initial studies on analytical application of these oligonucleotide conjugates are discussed.     

New Strategies for the Chemical Synthesis of Biologically Important Nucleic Acid Derivatives

Abstract

This paper describes general methods for the synthesis of N-phosphorylated ribonucleosides and oligonucleotides containing a 2′-O-phosphorylated or 2′-O-thiophosphorylated ribonucleoside. The NMR-based conformational analysis and computational molecular dynamics simulation of the 2′-O-phosphorylated ribonucleoside residue in such modified oligonucleotides suggested that the ribose residue existed preferentially in a C2′-endo conformation. It was also found that simple heating of 2′-O-phosphorylated oligonucleotides resulted in rapid dethiophosphorylation.     

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels-Alder Bioconjugation

Abstract

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

Synthesis and Incorporation of 2′-O-Methyl-Pseudouridine into Oligonucleotides

Abstract

A short multigram synthesis of 2′-O-methylpseudouridine and its phosphoramidite derivative is described which avoids the use of protecting groups on the nitrogens. A binding study of oligonucleotides containing this modification suggest an increased binding affinity to RNA when compared to oligonucleotides incorporating 2′-O-methyluridine.     

Intercalating Insert into Internucleotide Linkages as Way for Stabilization and Detection of of Short DNA Duplexes

Abstract

Simple and convenient method for stabilization and detection of duplexes of short oligonucleotides with DNA was developed. This method is based on use of oligonucleotides containing inercalating insert in internucleotide linkage. The linker is so long that dye can intercalate only into the same stacking contact. Additionally the method allows to introduce into oligonucleotide as one intercalator as well as several identical or different intercalating dye.     

Boranophosphate Oligonucleotides: New Synthetic Approaches

Abstract

Recently our laboratory reported a new backbone-modified class of oligonucleotides, with a borane (B₃3?) group replacing one of the non-bridging oxygen atoms. Here we present two new approaches to synthesize the boranophosphate oligonucleotides. All-stereoregular boranophosphate oligonucleotides can be prepared by enzymatic template extension reactions using nucleoside a-boranotriphosphates, which are good substrates for a number of polymerases. Larger scale synthesis of boranophosphate oligonucleotides can be carried out by effective chemical synthesis using the H-phosphonate approach, instead of previously used phosphoramidite methodology. The main advantage of H-phosphonate methodology is the ability to carry out one boronation reaction, after oligonucleotide chain elongation has been completed, using mild conditions without base damage and producing the desired boranophosphate oligonucleotides in high yield.     

Synthesis and Properties of New Fluorescein-Labeled Oligonucleotides

Abstract

2,4-Dinitroaniline is an efficient intramolecular fluorescence-quencher for fluorescein - labeled oligonucleotides and interacts with the heterocyclic bases on duplex formation. Consequently, intramolecular fluorescence quenching is disturbed in double labeled oligonucleotides of this type, and fluorescein shows strong fluorescence in a duplex form. There is a substential increase of the fluorescence-quantum yield when the marker and quencher is attached to a single guanosine residue. Two kinds of doubly labeled oligonucleotides have been synthesized, using the NPE/NPEOC strategy.