Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm / 中国应用生理学杂志

Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.

Mechanism of hydrogen treatment for cancer induced by oxidative damage / 肿瘤

As a new type of medical gas, hydrogen has therapeutic effect on many diseases. Studies have shown that the development of cancer is closely related to oxidative stress, which can damage the chromosomes, bases, proteins and lipids. Hydrogen molecules, as reducing substances, can alleviate the damage and protect cell function by inhibiting the signal transduction of cancer throu gh signal transduction. It provides a new research direction for treatment of cancer.

Structure and function of c-di-GMP riboswitches / 生物工程学报

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous nucleotide second messenger present in a wide variety of bacteria. It regulates many important bacterial physiological functions such as biofilm formation, motility, adhesion, virulence and extracellular polysaccharide synthesis. It binds with many different proteins or RNA receptors, one of which is called riboswitch that is usually located at the 5'-untranslational region (5'-UTR) in some mRNA. Riboswitch usually comprises a specific ligand-binding (sensor) domain (named aptamer domain, AD), as well as a variable domain, termed expression platform (EP), to regulate expression of downstream coding sequences. When a specific metabolite concentration exceeds its threshold level, it will bind to its cognate riboswitch receptor to induce a conformational change of 5'-UTR, leading to modulation of downstream gene expression. Two classes of c-di-GMP-binding riboswitches (c-di-GMP-Ⅰ and c-di-GMP-Ⅱ) have been discovered that bind with this second messenger with high affinity to regulate diverse downstream genes, underscoring the importance of this unique RNA receptor in this pathway. Class Ⅰ c-di-GMP riboswitches are present in a wide variety of bacteria, and are most common in the phyla Firmicutes and Proteobacteria, while class Ⅱ c-di-GMP riboswitches typically function as allosteric ribozymes, binding to c-di-GMP to induce folding changes at atypical splicing site junctions to modulate downstream gene expression. This review introduces the discovery, classification, function, and also the affected downstream genes of c-di-GMP riboswitches.

Effect of the PGE_2 Combined with TNF-? to the IP3-Ca~(2+) Pathway Mediated by Membrane TNF-?R of Fibroblasts / 环境与健康杂志

Objective To study the effect of different prostaglandin E2 (PGE2) combined with tumor necrosis factor-?(TNF-?) to the IP3-Ca2+ pathway mediated by membrane TNF-?R of fibroblasts. Methods The lung fibroblasts of breed mouse were primarily cultured. 10 ?g/L TNF-? and 10 ?g/L TNF-? combined with PGE2 of different doses were added to culture medium of fibroblasts. At some observation time,the expression of TNF-?R on the cellular membrane of fibroblasts was detected by the method of immunohistochemistry,the level of IP3 in cells was detected by the method of radioimmunoassay and the contents of Ca2+ in fibroblasts by the method of flow cytometry. Results With doses of PGE2 increasing at 30 s time point and 60 s time point,the cpm value of IP3 of fibroblast decreased gradually. The cpm value of IP3 significantly increased when the dose of PGE2 was 1.0 ?g/L,but obviously lower when the dose of PGE2 was 2.0 ?g/L at 120 s time point. The expression of TNF-?R and the contents of Ca2+ decreased in a dose-dependent way with the dose of PGE2 increasing. Conclusion Certain dose of PGE2 could suppress the effect that TNF-? enhance the cell proliferation of fibroblast by the IP3-Ca2+ pathway mediated by membrane TNF-?R of fibroblasts.

Modulation of Large Conductance Ca2+-activated K+ Channel of Skin Fibroblast (CRL-1474) by Cyclic Nucleotides

Potassium channels in human skin fibroblast have been studied as a possible site of Alzheimer disease pathogenesis. Fibroblasts in Alzheimer disease show alterations in signal transduction pathway such as changes in Ca2+ homeostasis and/or Ca2+-activated kinases, phosphatidylinositol cascade, protein kinase C activity, cAMP levels and absence of specific K+ channel. However, little is known so far about electrophysiological and pharmacological characteristics of large-conductance Ca2+-activated K+ (BKCa) channel in human fibroblast (CRL-1474). In the present study, we found Iberiotoxin- and TEA-sensitive outward rectifying oscillatory current with whole-cell recordings. Single channel analysis showed large conductance K+ channels (106 pS of chord conductance at +40 mV in physiological K+ gradient). The 106 pS channels were activated by membrane potential and [Ca2+]i, consistent with the known properties of BKCa channels. BKCa channels in CRL-1474 were positively regulated by adenylate cyclase activator (10microM forskolin), 8-Br-cyclic AMP (300microM) or 8-Br-cyclic GMP (300microM). These results suggest that human skin fibroblasts (CR-1474) have typical BKCa channel and this channel could be modulated by c-AMP and c-GMP. The electrophysiological characteristics of fibroblasts might be used as the diagnostic clues for Alzheimer disease.

The Regulation of AP-1 DNA Binding Activity by Long-term Nicotine Stimulation in Bovine Adrenal Medullary Chromaffin Cells: Role of Second Messengers

The signal pathways involved in the regulation of AP-1 DNA binding activity in long-term nicotine stimulated bovine adrenal medullary chromaffin (BAMC) cells have not been well characterized. To understand the involvement of second messengers in the regulation of AP-1 DNA binding activity, the present study was designed to define the time-course for inhibition of nicotine-induced responses by cholinergic antagonists, Ca2+ and calmodulin (CaM) antagonists, and calcium/calmodulin-dependent protein kinase (CaMK) II inhibitor using electrophoretic mobility shift assay. Nicotine (10microM) stimulation increased AP-1 DNA binding activity at 24 hr after treatment. Posttreatment with hexamethonium (1 mM) plus atropine (1microM) (HA), nimodipine (1microM), or calmidazolium (1microM) at 0.5, 3, and 6 hr after the nicotine treatment significantly inhibited the AP-1 DNA binding activity increased by long-term nicotine stimulation. However, posttreatment with HA, nimodipine, or calmidazolium at 9 or 12 hr after the nicotine treatment did not affect the nicotine-induced increase of AP-1 DNA binding activity. The pretreatment of BAMC cells with various concentrations of KN-62 inhibited the increase of AP-1 DNA binding activity induced by nicotine in a concentration-dependent manner. KN-62 (10microM) posttreatment beginning at 0.5, 3, or 6 hr after the nicotine treatment significantly inhibited the increase of AP-1 DNA binding activity. However, KN-62 posttreatment beginning at 9 or 12 hr after the nicotine treatment did not affect the increase of AP-1 DNA binding activity. This study suggested that stimulation (for at least 6 hr) of nicotinic receptors on BAMC cells was necessary for increase of AP-1 DNA binding activity, and activation of Ca2+, CaM, and CaMK II up to 6 hr at least seemed to be required for the increase of nicotine-induced AP-1 DNA binding activity.

The Effects of Chronic Carbamazepine Administration on Protein Kinase A and Protein Kinase C Activities in Rat Brain

OBJECTIVE: Many evidences suggest that patients with bipolar disorder have functional abnormalities in their postreceptor signal transduction pathways, and mood stabilizing effect of lithium is exerted by modulating this dysfunctioning system. Carbamazepine, an antiepileptic agent, is also known to be effective in the treatment and prevention of bipolar disorder. But the precise mechanism of action of the drug is still poorly understood. This study was performed to elucidate the possible therapeutic mechanism of carbamazepine. METHOD: The effects of chronic carbamazepine administration on protein kinase A and protein kinase C activities in frontal cortex of rat brain after 2 weeks of drug administration were measured and compared with those of control subjects. RESULTS: Mean(+/-SE) value of activity(phosphate transfer micromol/mg of protein, min) or protein kinase A in control and test group was 0.249563+/-0.036 and 0.539853+/-0.078, and that of protein kinase C was 0.654817+/-0.053 and 1.146205+/-0.052 respectively, being increased in test group. And differences between the two groups were statistically significant for both enzymes(protein kinase A ; p<0.01, protein kinase C ;p<0.001). CONCLUSION: These results show that chronic carbamazepine administration increases protein kinase A and C activities, and concerning the possible mode of therapeutic action in bipolar disorder it is suggested that enhanced enzymes phosphorylate receptor-G-protein-effector complexes to dampen hyperfunctioning neuronal activity and thus stabilize the system.

Mecanismo de acción de los antidepresivos: una mirada histórica / Mechanism of action of antidepressants: a historical look

En la década del 60, se asumía que la depresión consistía en una deficiencia de catecolaminas (hipótesis catecolaminérgica) y que los antidepresivos tricíclicos actuaban sobre ellas (principalmente noradrenalina) incrementándolas o potenciándolas a nivel central. Otras teorías sobre las bases biológicas de la depresión y el mecanismo de acción de los antidepresivos fueron planteadas posteriormente a medida que se daban evidencias sobre la participación de otros neurotransmisores. En la actualidad, se asume que el tipo de neurotransmisor implicado no es tan importante como los sistemas intraneuronales de traducción y transcripción de señales activados por la acción de los antidepresivos y que permiten estimular los mecanismos homeostáticos alterados de las neuronas disfuncionales, produciendo adaptaciones terapéuticas que llevan a alteraciones sustanciales y duraderas en la función neurona I y por lo tanto, a un nuevo estado funcional...

In the decade of6Os, were assumed that the depression was consistíng of a deficiency of catecholamines (catecholaminergic hypothesis) and that the trícyclic antidepressants were acting on them (mainly norepinephrine) increasing them at central level. Other theories on the biológical bases of depression and the actíon mechanism of antidepressants were outlined later while were given evidence on the participation of others eurotransmitters. Atpresent, itis assumed that the type of neurotransmitter involved is not so important as the intraneuronal systems ofsigns translation and transcription activated by the action of antidepressants and that permit to stimuíate homeostatic mechanisms alterad of disfunctional neurons, producing therapeutic adjustments that carry to substantial and lasting alterations in the neuronal function and there fore, to a new functíonal state...

Pharmacological and molecular biological characteristics of store-operated calcium ciannels / 中国药理学通报

Store operated calcium channels (SOCCs) are referred to as plasma membrane calcium channels that are opened in response to a depletion of intracellular calcium stores. SOCCs are broadly distributed in excitable and non excitable cells, and may play important roles in conducting intracellular calcium signals, modulations of cell functions and gene expression. Although the current understanding of SOCCs related mechanism is still limited, the physiological significance of SOCCs and their possible links to certain diseases have been suggested. Future study of highly selective SOCCs modulating agents may promote not only related research, but also the development of novel therapeutic drugs.

Cross—linking Membranous CD23 Molecules on B Lymphocytes Affect Its Proliferation and Second Messenger / 中国免疫学杂志

The possible roles of CD23 molecule on B cells were analyzed through cross—linking CD23 molecules on tonsillar B lymphocytes with CD23 natural ligands IgE and anti—CD23 McAb.After cross—linking CD23 molecules on activated B cells,the change of B cell proliferation and second messengers were observed.The results showed:(1)Double—valence anti—CD23 McAb and multi—valence IgE immune complex(IgEIC)had a two—way effect on B cellsproliferation,on high concentrations,they inhibited the proliferation of a etivated B cells by sAo In con trast with a certain lower concentration,they promoted the proliferation of activted B cells.The two—way effect depended on IL—4 which functions to up—regulate CD23;(2)mono —valence IgE had no observeable effects on B cells proliferation;(3)IgEIC and antl—CD23 McAb at the same concentration as stimulating B cell proliferation increased the intracellularCAMP levls,but not cGMP levels.(4)Anti—CD23 McAb provoked the transent increase of in tracellular free(Ca~(2+)).

Ihe Effect of Arginine Vasopressin on Acute Ischeme Brain Edema and Its Mechanism / 第二军医大学学报

The purpose of this experiment was to study the role of arginine vasopressin (AW) in acute cerebral ischemic edema in mongolian gerbils. The results showed that intracerebroventricular injection (ICV) of AVP exacerbated the ischemic brain edema, while ICV of AW antiserum significantly decreased the ischemic brain edema. Nimodipine couldn't block this role of AW in ischemic brain edema. The cortical Na+ -K+ ATPase activity was significantly decreased, the contents of cAMP in the ischemic cortex and hypothalamus and the contents of cGMP in the hypothalamus were remarkably increased after ICV of AW. These suggest AW was involved in the pathophysiologic process of acute ischemic brain edema. And its mechanism might be the effect of AW on AW receptor mediated by cAMP, cGMP, and that in turn inhibited the Na+ -K+ ATPase activity of brain cell membrane, then exaggerated the formation of ischemic brain edema.