Electroacupuncture improves neuropathic pain Adenosine, adenosine 5'-triphosphate disodium and their receptors perhaps change simultaneously

Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 （P2X3） receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.

Changes in P2Y purinoreceptor-mediated intracellular calcium signal pathways results in inositol-1, 4, 5-triphosphate-sensitive calcium stores in rat small trigeminal ganglion neurons

BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Two pathways are involved in the purinoreceptor-mediated [Ca^2＋]i rise observed in nociceptive trigeminal ganglion neurons. One pathway involves the metabotropic P2Y receptors, which are associated with the IP3 sensitive Ca^2＋store, and the second pathway is coupled to ionotropic P2X receptors that induce the Ca^2＋ influx.

Commentary Potential Enhancement by 3-Deazauridine of the Antiviral Activity of Molnupiravir in Patients with COVID-19

Background: Molnupiravir, N4-hydroxycytidine-5’-isopropyl ester, is an oral prodrug of N4-deoxycytidine (NHC), a nucleoside analog, which has in vitro activity against SARS-CoV-2. NHC is phosphorylated in cells to NHC triphosphate (NHC-TP), which is incorporated into viral RNA, leading to epigenetic catastrophe of the viral genome and inhibition of viral replication. The antiviral activity against SARS-CoV-2 is dependent on the number of molecules of NHC-TP incorporated into viral RNA. Clinical studies in patients with COVID-19 showed that treatment with molnupiravir for 5 days decreases the risk of hospitalization and death as compared with placebo. Objective: It should be possible to enhance the antiviral activity of NHC-TP against SARS-CoV-2 by the use of the biochemical modulator, 3-deazauridine (3DU). 3DU is an inhibitor of CTP synthetase. Inhibition of this enzyme results in a reduction in the intracellular pool size of CTP. Since NHC-TP competes with CTP for incorporation into viral RNA in the reaction catalyzed by the SARS-CoV-2 viral RNA-dependent RNA polymerase, the reduction in the level of CTP should result in a significant enhancement of the incorporation of NHC-TP into viral RNA and an enhancement of its antiviral activity. Methods: Analysis of the publications of 3DU and cytosine nucleoside analogues support the hypothesis that 3DU enhances the pharmacological action of the analogues. Results: 3-DU increased the incorporation of 5-azacytidine into RNA and 5-aza-deoxycytidine into DNA of leukemic cells with an enhancement of their antineoplastic action. 3-DU potentiated the antiviral activity against HIV-1 activity by the cytosine nucleoside analogues: 2’-deoxy-3’-thiacytidine (3TC;lamivudine) and 2’,3’-dideoxycytidine (ddC). This anti-HIV-1 activity of 3DU was associated with a reduction in the intracellular pool size of dCTP and increased incorporation of triphosphates of 3TC and ddC into DNA by the HIV-1 reverse transcriptase. The reduction of CTP levels in cells by 3-DU also leads to a reduction in dCTP since CTP is its precursor. Conclusion: The preclinical studies on 3-DU indicate that it can enhance the pharmacological activity of both ribo- and deoxyribonucleoside analogues against neoplastic cells and viral infected cells. These observations suggest that 3-DU also has the potential to enhance the antiviral activity of molnupiravir and arrest the progression of the disease in patients with COVID-19.

The Chemical Synthesis of 4'-Thio-2'-deoxythymidine-5'-triphosphate and Its Effects on DNA Synthesis

Scientists have long been interested in the synthesis and medical use of 4’-thionudeosides. However, their effect on DNA synthesis has not been well studied. In order to study their bioactivity and explore new applications, we have synthesized 4’-thio-2’-deoxythymidine-5’-triphosphate (T’TP). The experimental results obtained in our lab showed that T’TP is a strong inhibitor of DNA polymerase, and the inhibition is highly specific. These prbperties indicate the potential of T’TP used for antitumor and antiviral agents as well as for the terminator in DNA sequencing.

Spectrofluorometry of Ions and Small Molecules Based on Conformational Changes of Specific Oligonucleotides with o-Phthalaldehyde-β-mercaptoethanol

Specific oligonucleotides such as telomere DNA and aptamer often undergo conformational changes upon ligand binding. Composite reagent composed of o-phthalaldehyde and β-mercaptoethanol（OPAME） has been extensively applied to fluorescent detection of amino compounds based on the reaction of primary amino-group, herein we proposed a general spectrofluorometry for ions and small molecules due to conformational changes upon ligand binding taking K^＋ and ATP as examples. In a borate controlled buffer medium, telomere DNA could react with OPAME, giving a thio-subtituted isoindole compound with strong fluorescence emission at 455 nm when excited at 340 nm. It was found that however, the fluorescence emission was greatly reduced in the presence of K^＋ since the formation of the quadruplex structure inhibits the reaction activity of amino-groups of telomere DNA. In order to testify the general application of OPAME reagent based on the conformational change of oligonucleotides, we further proposed a sensitive method of ATP based on its highly selective interaction with ATP-aptamer. The above mentioned applications show that the spectrofluorometry with the aid of OPAME reagent is simple, label free that is expected to be potentially general for DNA conformational change-based target detection.

Assessing the Adverse Impacts of PM_(2.5)on Olfactory System Using an Air-liquid Interface Culture Model of Primary Olfactory Epithelial Cells

The air-liquid interface(ALI)culture is a kind of recently developed system,which has proved its availability in simulating the biology of respiratory tract epithelial tissues.In this study,an ALI-based mouse primary olfactory epithelial cell(OEC)model was established to perform the exposure of PM_(2.5)(PM=particulate matter)collected from Dianshan Lake(Shanghai)and Wangdu(Hebei).The results showed that PM_(2.5)in both regions caused a decrease in cell viability in a dose-dependent manner.The 0.5 and 5µg/cm^(2)(around ambient concentrations)of PM_(2.5)disrupted OEC membrane integrity and produced oxidative stress with elevated indicators of malondialdehyde(MDA)and reactive oxygen species(ROS).In transcriptomic sequencing,the terms concerning inflammatory cytokines and second messenger cyclic adenosine-3′,5′-monophoshate(cAMP)were enriched in two treatments.The cytokine array showed the levels of some cytokines were altered,although inflammatory responses may not remarkably occur.Meanwhile,PM_(2.5)disturbed cAMP contents and key genes in the cAMP signaling pathway.The effects of PM_(2.5)of both regions were largely consistent,while Wangdu samples caused more ROS and Dianshan Lake samples tended to induce inflammatory injury.Thus,with the application of a novel ALI-based in vitro OEC model,our study demonstrated that ambient PM_(2.5)has the ability to threaten the physiologies and functions of the olfactory system.

STUDY ON THE MECHANISM OF INTERFERON ACTION (Ⅺ)——EFFECT OF pppA2'p5'A2'p5'A ON THE LEVEL OF cAMP AND cGMP IN MACROPHAGES

In this paper, the increase of cellular cAMP and cGMP levels in macrophages induced bypppA2’p5’A2’p5’A （briefly 2’-5’P3A3） is first reported. The optimal concentration of 2’-5’P3A3 for the elevation of cellular cGMP to the highest level is 10-7-10-6mol/L, while thatfor cAMP is 10-7mol/L. The time for cGMP to reach its peak value is 15 min and that forcAMP is 2 h, when the cells are treated with 2’-5’ P3A3 at 10-7mol/L, which is the optimalconcentration for developing biological effect of macrophages （phagocytosis）. These resultssuggest that cGMP and cAMP may be related to, or may be the mediators for, 2’-5’P3A3action.

Spectrofluorimetric Study on the Weak Interaction between ATP and Na-4-Tosyl-L-arginine Methyl Ester Hydrochloride

In this paper, some new results on the selective weak interaction between Na-4-tosyl-L-arginine methyl ester hydrochloride （TAME） and adenosine-5＇-triphosphate （ATP） have been reported. Fluorescence spectrophotometry and Fourier transform infrared （FT-IR） spectroscopy were used to investigate this kind of weak interaction. In fluorescence experiments, obvious fluorescence quenching phenomena were observed when TAME was added, which indicated the weak interactions between TAME and ATP. It has been identified by fluorescence titration experiments that TAME exhibited high selectivity to ATP over ADP and AMP. FT-IR spectral results showed that an ATP-TAME adduct was formed. The experimental results indicated that the interaction sites were the guanidinium group of TAME main-chain and the γ-phosphate group of ATP, and the interaction took place through hydrogen bonding and electrostatic force. In addition, the effects of metal ions on the weak interaction between ATP and TAME, or between ATP and analogues of L-arginine were studied.

Sperm glyceraldehyde 3-phosphate dehydrogenase gene expression in asthenozoospermic spermatozoa

It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.

Fluorescent probes for recognition of ATP

Adenosine 5＇-triphosphate（ATP） not only participates in various physiological activities as the universal energy currency but also implicates in various pathological processes in living cells. Consequently,sensitive and selective detection ATP in live cells, tissues, as well as environmental samples, are urgently demanded. Due to the simple and convenient operation, economy cost, high selectivity for analyte, well biocompatibility and low cytotoxicity, fluorescent sensors for monitoring ATP have aroused great attention of researchers. In recent years, a large number of fluorescent sensors for detecting ATP have developed. This manuscript summarized most of these sensors and the interaction-mechanism between ATP and sensors, mainly including electrostatic interaction, p-p interaction, covalent bonding or hydrogen bond, or combinations of them, and the advantages of each strategy were also generalized.Here, a viewpoint of classification was shown where the sensors were divided into five typed ones according to the structure of probes used.