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.

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.

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.