ABSTRACT
Licking behavior is important for water intake. The deep mesencephalic nucleus (DpMe) has been implicated in instinctive behaviors. However, whether the DpMe is involved in licking behavior and the precise neural circuit behind this behavior remains unknown. Here, we found that the activity of the DpMe decreased during water intake. Inhibition of vesicular glutamate transporter 2-positive (VGLUT2+) neurons in the DpMe resulted in increased water intake. Somatostatin-expressing (SST+), but not protein kinase C-δ-expressing (PKC-δ+), GABAergic neurons in the central amygdala (CeA) preferentially innervated DpMe VGLUT2+ neurons. The SST+ neurons in the CeA projecting to the DpMe were activated at the onset of licking behavior. Activation of these CeA SST+ GABAergic neurons, but not PKC-δ+ GABAergic neurons, projecting to the DpMe was sufficient to induce licking behavior and promote water intake. These findings redefine the roles of the DpMe and reveal a novel CeASST-DpMeVGLUT2 circuit that regulates licking behavior and promotes water intake.
ABSTRACT
<p><b>OBJECTIVE</b>To construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.</p><p><b>METHODS</b>We firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.</p><p><b>RESULTS</b>Our results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.</p><p><b>CONCLUSION</b>Our protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.</p>
Subject(s)
Humans , Cloning, Molecular , DNA, Complementary , Genetics , Escherichia coli , Genetics , Genetic Vectors , Histones , Genetics , Polymerase Chain Reaction , MethodsABSTRACT
<p><b>AIM</b>To explore the effect of hyperhomocysteinemia on vascular calcification and the underlying mechanism of it.</p><p><b>METHODS</b>Arterial calcification of Sprague-Dawley rats was induced by vitamin D3 plus nicotine. Hyperhomocysteinemia was established by feeding high methionine diet for six weeks and was assessed b y plasma total homocysteine (tHcy) level detected by HPLC method. Calcification was confirmed by von Kossa staining, measurement of calcium content, alkaline phosphatases (ALP) activity and osteocalcin (OC) concentration of vascular tissue. Lipid conjugated dienes formation were determined to reflecting the production of lipid peroxide.</p><p><b>RESULTS</b>The results showed that there were mass black granules deposited in aortic wall of the calcified rats by von Kossa staining. Calcium content, ALP activity, OC concentration in calcified rats increased by 8.09-fold, 45.57% and 2.81-fold compared with those of the control group (P < 0.01). Calcium content in calcified rats with high methionine diet increased by 34.29% compared with that of the calcified rats, while ALP activity and OC content decreased by 29.13% and 74.69% compared with that of the calcified rats. Lipid conjugated dienes formation in plasma of the rat with high methionine diet and of calcified rats with high methionine diet increased by 1.93 and 2.89-fold compared with those of the control group, respectively (P < 0.01), and in calcified rats with high methionine diet group was increased by 32.90% compared with that of high methionine diet group (P < 0.01).</p><p><b>CONCLUSION</b>Hyperhomocysteinemia could promote vascular calcification, which might be mediated through the production of lipid peroxide.</p>