In Situ Bioorthogonal Metabolic Labeling for Fluorescence Imaging of Virus Infection In Vivo.

Optical fluorescence imaging is an important strategy to explore the mechanism of virus-host interaction. However, current fluorescent tag labeling strategies often dampen viral infectivity. The present study explores an in situ fluorescent labeling strategy in order to preserve viral infectivity and precisely monitor viral infection in vivo. In contrast to pre-labeling strategy, mice are first intranasally infected with azide-modified H5N1 pseudotype virus (N3 -H5N1p), followed by injection of dibenzocyclooctyl (DBCO)-functionalized fluorescence 6 h later. The results show that DBCO dye directly conjugated to N3 -H5N1p in lung tissues through in vivo bioorthogonal chemistry with high specificity and efficacy. More remarkably, in situ labeling rather than conventional prelabeling strategy effectively preserves viral infectivity and immunogenicity both in vitro and in vivo. Hence, in situ bioorthogonal viral labeling is a promising and reliable strategy for imaging and tracking viral infection in vivo.

[Characteristics in the feature of 3H-Ryanodine binding to cardiomyocyte nuclei in reperfusion injury in rat].

OBJECTIVE: To investigate the changes in binding features of (3)H -Ryanodine cardiac myocytic nuclei in reperfusion injury in the rat, and the effects of phosphorylation regulation on the binding characteristics. METHODS: Healthy male Wistar rats were randomly divided into ischemia/reperfusion injury (IRI) group and sham-operation group. IRI model was reproduced by ligating the left main coronary artery for 30 minutes followed by reperfusion of the heart for 3 hours, while in the sham-operation group the animals underwent a thoracotomy only for 3.5 hours. Cardiac myocytic nuclei were isolated by sucrose density gradient centrifugation. The maximum binding capacity (Bmax) and the dissociating ratio (Kd) of Ryanodine receptors (RyRs) were measured by radioligand binding analysis as well as Scatchard plot. RESULTS: There was a high affinity of RyRs to bind with (3)H-Ryanodine on the nuclear wall of rat cardiac myocytic nucleus. Compared with that of the sham-operation group, Bmax of RyRs of IRI cardiac myocytic nuclei was decreased by 29% (P<0.01), but there was no difference in Kd between two groups(P>0.05). With phosphorylation by activating the endogenous protein kinase C(PKC) with phorbol myristate acetate (PMA)+phosphatidyl serine(PS), Bmax of both sham and IRI groups were increased markedly (P<0.01), but in the latter group it was less increased (P<0.01). With phosphorylation by Ca(2+)-calmodulin (CaM), Bmax was decreased in both sham-operation and IRI groups (both P<0.05), but was less decreased in the latter(P<0.01). However, both PMA+PS and Ca(2+)-CaM did not change the Kd of nuclear RyR in either group (both P>0.05). CONCLUSION: After IRI, Bmax of (3)H -Ryanodine of cardiac myocytic nuclei is decreased and the impact of PMA+PS and Ca(2+)-CaM on the Bmax is impaired, but the affinity of (3)H -Ryanodine to cardiac myocytic nuclei is not altered under above circumstances.

[Change of cardiac myocyte nuclear inositol 1,3,4,5-tetrakisphosphate receptor binding properties in rat with myocardial ischemic reperfusion].

OBJECTIVE: To observe the alteration of cardiac myocyte nuclear inositol 1,3,4,5-tetrakisphosphate receptor (IP(4)R) binding properties in rat subjected to myocardial ischemic reperfusion in order to further make it clear whether this change is involved in the molecule mechanism of cell apoptosis of rat with myocardial ischemic reperfusion. METHODS: Extracting of cardiac myocyte nucleus was accomplished by saccharose density gradient centrifugation method, the binding properties of nuclear IP(4)R in different conditions were detected by radioligand binding assay. Apoptosis index of myocardial cell was determined by using TUNEL assay. RESULTS: (1) Myocardial cell apoptosis index in rat heart underwent 30 min regional ischemia and 3 h reperfusion increased distinctly compared with that in control group (P < 0.01). (2) There were two IP(4) binding sites located to the nuclear envelope. (3) In ischemic reperfusion injury (IRI) group, Bmax from high affinity binding site of nuclear IP(4)R significantly increased compared with that in sham-operated group, whereas Bmax from low affinity binding site didn't change. Kd values of both sites were all significantly decreased by 63% and 55%, respectively. (4) Phosphorylation of nuclear IP(4)R by PKC increased markedly its binding ability both in IRI and control group (P < 0.05), which was more apparent in IRI group. (5) In sham-operated group, the binding ability of nuclear IP(4)R increased with increasing free calcium concentrations in cytoplasm, and the binding properties of IP(4)R in IRI group were also increased in the condition of calcium overloading. CONCLUSION: The increasing of binding properties of nuclear IP(4)R from ischemic reperfusion heart may be one of important mechanism involved in myocardial cell apoptosis, furthermore resulting in myocardial IRI.

[Regulatory effects of nucleotides on ATP-sensitive potassium channel opener pinacidil-induced vascular relaxation].

AIM: In order to evaluate the regulatory effects of nucleotides and adenosine on ATP-sensitive potassium channel (K(ATP)) in artery smooth muscles, the effects of them on vascular relaxation induced by K(ATP) opener pinacidil(Pin) were investigated. METHODS: The isolated endothelium- denuded aorta rings were preincubated with nucleotides or nucleotides and glibenclamide (Gli) for 10 min, the vascular relaxation induced by Pin in aorta precontracted with 20 mmol x L(-1) KCl was observed. RESULTS: After the isolated endothelium-denuded aorta rings were preincubated with ATP, ADP, UDP, GTP and adenosine (Ade) 100 micromol x L(-1) respectively, the vascular relaxation induced by Pin was changed as following: (1) ATP could inhibit the K(ATP) activation by Pin and enhance the blockade of K(ATP) by Gli. (2) ADP could inhibit the K(ATP) activation by Pin and attenuate the blockade of K(ATP) by Gli. (3) The regulatory effect of Ade on K(ATP) was similar with that of ADP. (4) UDP could enhance the K(ATP) activation by Pin and attenuate the blockade of K(ATP) by Gli. (5) GTP could enhance the K(ATP) activation by Pin, but had no effects on the blockade of K(ATP) by Gli. CONCLUSION: Nucleotides and adenosine, related to energy metabolism, could modulate the functions of K(ATP) in vascular smooth muscle. But their pharmacological characteristics were different.

Alteration of binding sites for [3H]P1075 and [3H]glibenclamide in renovascular hypertensive rat aorta.

AIM: The alterations of the binding sites for ATP-sensitive K+ channel (K(ATP)) openers and blockers in aortic strips were investigated in hypertensive rats. METHODS: Radioligand binding techniques were used to compare the specific binding properties of [3H]P1075 and [3H]glibenclamide (Gli) in normotensive (NWR) and reno-vascular hypertensive rat (RVHR) aortic strips. RESULTS: The KD values of [3H]P1075 binding were increased by 1.5-fold, while the Bmax values were unchanged in RVHR. The IC50 values of P1075 and pinacidil (Pin) for displacing the [3H]P1075 binding in RVHR were increased by 1.8- and 1.7-fold, respectively. The kinetic processes of association and dissociation of [3H]P1075 binding were slower in RVHR. Glibenclamide pretreatment slowed down the kinetic processes of the association and dissociation of [3H]P1075 binding in NWR, but failed to alter the kinetic processes of [3H]P1075 binding in RVHR. The IC50 values of Gli for displacing the [3H]Gli binding at high-affinity sites were increased by 3-fold, while those at low-affinity sites remained to be unchanged in RVHR. The kinetic processes of association of [3H]Gli binding were decreased and those of the dissociation were accelerated in RVHR. The treatment with Pin slowed down the association kinetic processes but accelerated the process of the dissociation of [3H]Gli binding in NWR, but did not alter the kinetics of [3H]Gli binding in RVHR. CONCLUSION: The affinity of binding sites for [3H]P1075 and of high-affinity binding sites for [3H]Gli are decreased, and the negative allosteric interactions between the two binding sites are impaired in RVHR aorta.