Upregulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin-enhanced green fluorescent protein.

Upregulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin-enhanced green fluorescent protein.

Furosemide, used worldwide as a diuretic agent inhibits sodium reabsorption in the henle loop, resulting in diuresis and natrisis. Arginine Vasopressin (AVP) is synthesized in the suprooptic core (wire), paramencentricular core (PVN) and suprachiasmatic core (SCN) of the hypothalamus. Synthesis AVP in the magnocyal neurons of physiologically regulated physiologically regulated PVN by plasma osmoleality and blood volume and contributed to the homeostasis of water by increasing the reabsorption of water in the collection conduit. The dynamics of central AVP after the peripheral administration of furosemide remains uncertain.

Here we have studied the effects of the intraperitoneal administration (IP) of furosemide (20 mg / kg) on ​​hypothalamic AVV using transgenic rats expressing a green fluorescent protein improved by the AVP (EGFP) under the AVP promoter . I.P. The administration of furosemide has not affected plasma osmoleality in this study; However, EGFP in the PVN’s magnocular son and divisions (MPVN) have been significantly increased after the administration of furosemide compared to control. Immunohistochemical analysis revealed FOS (IR) type immunoretivity in EGFP positive neurons in the son and MPVN 90 min after I.P. The administration of furosemide and heteronuclear AVP (HN) RNA and EGFP mRNA levels have been significantly increased. These changes induced by furosemide have not been observed in Suprachiasmatic AVP neurons.

In addition, furosemide has induced a remarkable increase in FOS-IR in the terminals of laminae vasculosum organum (OVLT), median preoptic core (MNPO), submissive organ (SFO), locus coauauus (LC), solitary tract core ( NTS), and Ventrolateral Rostral Medululla (RVLM) after IP Administration of Furosemide. In conclusion, we were able to visualize and quantitatively evaluate the synthesis of AVP-EGFP and neuronal activations after the peripheral administration of furosemide, using the AVP-EGFP transgenic rats. The results of this study can provide new perspectives on the elucidation of the physiological mechanisms underlying the corporal fluid homeostasis induced by furosemide. This article is protected by copyright. All rights reserved.

 

Transpondable mediation assembly of the fluorescent protein removed tripartite for the assay without label of the activity of the output.

Transpondation of surface proteins catalyzed by transpideptidase plays a crucial role in the process of infection of the gram-positive pathogen and the activity of activity and the screening of its inhibitors is of great importance for biological research. and fundamental pharmaceutical development, especially new antivirulence drugs. In this document, we have developed a new fluorescent biosensor to detect the activity of the tricase based on a triggered assembly on the transverse of green fluorescent proteins divided tripartite (divided GFP). PEPTIDE P1, composed the 10th GFP β sheet (GFP10) and the SLPTASE A (SRTA) and P2 P2 recognition sequence, the GFP 11th β-sheet (GFP11) with oligoglycin at N-terminal, were designed and synthesized, respectively.

The existence of active SRTA P1 and P2 to tackle a peptide, which could bind spontaneously to GFP1-9 (the first to the 9th sheets of the GFP) and assemble in the functional GFP. Thus, the catalyzed transpondation of the output can switch to the GFP fluorescence signal. The method has been successfully applied to detect the SRTA activity with a low detection limit of 0.16 nm and for its inhibition measurement. In addition, the feasibility of the proposed trial has also been expanded to detect SRTA in human blood and an analysis of gram-positive pathogens in frozen foods. Our method, using tripartite split GFP as reading, is easy, without label and sensitive and has great potential as a promising platform for the detection of morality and screening for inhibitors.

 Upregulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin-enhanced green fluorescent protein.
Upregulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin-enhanced green fluorescent protein.

Two distinct fluorescence states of the green fluorescent protein induced by the ligand.

Unag is a recently discovered ligand fluorescent protein that uses bound bilirubin (BR) as fluorophore. The fluorescence of the UNG-BR (Holounag) complex compares to the quantum efficiency with that of the improved green fluorescent protein, but it is superior in that the formation of fluorophore is instantaneous and non-oxygen dependent; As a result, great attention has been paid to UNAG as a new fluorescent probe. However, many important molecular properties of fluorescent probes remain unknown, such as BR association / dissociation rates, which determine the stability of it and the dispersibility of UNGUN in aqueous solutions, which influence functions. Marked proteins. In this study, we found, in the survey process on the association rate, that the Holounag takes two distinct fluorescence states, which we named Holounag1 and Holounag2.

The Holounag1 initially forms after the binding of BR then changes to the brightest holounag2 by a reversible intra-molecular reaction, thus reversing a balance between the two states. The spectroscopic analysis indicated that the intra-molecular reaction was not associated with a chemical change of Br but with a change in the environmental conditions surrounding the FR. We also revealed that the molecular brightness ratio and the two states’ equilibrium population ratio (Holounag1 / Holounag2) were 1: 3.9 and 6: 4, respectively, using a counting analysis of numbers. Photons.

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From these results, we suggested a new scheme, to our knowledge, for the formation of the Unag and BR complex system and determined the various interest rate constants they are associated with them. In addition, using an analytical ultracentrifugation, we have established that the UNGE in Apo-State (apounag) and the Holounag are monomer in aqueous solution. These results provide not only key information for the practical use of UNAG as a fluorescent probe, but also the possibility of developing a more brilliant genetic genetic genetic mutant at HOLOUNAG2 constituent.

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