TSPO ligand etifoxine attenuates LPS-induced cognitive dysfunction in mice.

The translocator protein (TSPO), once known as peripheral-type benzodiazepine receptor, was reported to be related with several physiological functions. Etifoxine is a clinically available anxiolytic drug, and has recently shown neuroprotective effects as a TSPO ligand. The aim of the present study was to investigate the influence of etifoxine on LPS-induced neuroinflammation and cognitive dysfunction. C57/BL6 male mice were injected with etifoxine (50 mg/kg, i.p.) three days before lipopolysaccharide (LPS, 500 µg/kg, i.p.) administration. Etifoxine pretreatment alleviated hippocampal inflammation, increased brain levels of progesterone, allopregnanolone and attenuated cognitive dysfunction in LPS-injected mice. While LPS increased expression of caspase-3 and decreased p-Akt/Akt, etifoxine returned caspase-3 and p-Akt/Akt to control levels. Finasteride, a 5α-reductase inhibitor that blocked allopregnanolone production, partially reversed the effects of etifoxine. We concluded that etifoxine exerted neuroprotective effects in LPS-induced neuroinflammation and the neuroprotection may be related with increase of neurosteroids synthesis and decrease of apoptosis.

Robotic-assisted laparoscopic artery-sparing varicocelectomy using indocyanine green fluorescence angiography: Initial experience.

We reported our initial experience of robotic-assisted laparoscopic artery-sparing varicocelectomy using indocyanine green (ICG) fluorescence angiography in treatment of varicocele. A total of 45 varicocelectomies in 27 patients were performed. The mean operation time was 49.1 ± 8.5 min for unilateral and 65.6 ± 8.3 min for bilateral repair. 47.2 s after ICG injection, testicular artery (TA) was visualised. After an interval of 31.3 s, fluorescent veins were identified. Of all the 45 spermatic cords, 68.9% had a solitary artery, while 31.1% had 2 arteries. The mean hospital stay was 1.6 ± 0.9 days. Semen concentration and motility were significantly improved 6 months after surgery, no recurrence, hydrocele or testicular atrophy was observed. Our study demonstrated that robotic-assisted laparoscopic artery-sparing varicocelectomy using ICG fluorescence angiography is a safe, effective and promising technique in treatment of varicocele.

Laparoscopic Yang-Monti Ureteral Reconstruction in Children.

OBJECTIVE: To investigate the clinical outcome of surgical treatment for long ureteral defect in children, we evaluated our experience of managing 6 children with the long defect utilizing laparoscopic ureteral reconstruction technique using Yang-Monti technique. MATERIALS AND METHODS: Six children with long ureteral defect who underwent laparoscopic Yang-Monti ureteral reconstruction between January 2013 and March 2016 were reviewed. The diagnosis and outcomes of long ureteral defects were reviewed based on clinical and imaging data. We assessed preoperative clinical data and outcomes, and analyzed the experience of laparoscopic Yang-Monti ureteral reconstruction. RESULTS: The mean age of the patients was 8.5 years. The etiology of the ureteral defect was failed pyeloplasty in 4 patients, failed pyeloplasty and ureteral reimplantation in 1, and trauma in 1. The mean defect length was 5.83 cm. All operations were performed successfully with no serious intraoperative complications and no conversion. The average operative time was 314 minutes, the average intraoperative blood loss was 25 mL, the average drain removal was 3.83 days, the average start of oral feeding was 5.17 days, and the average postoperative hospital stay was 7.17 days. Six patients suffered Clavien I and II complications postoperatively and were managed conservatively. Two patients suffered Clavien III complications postoperatively and were managed by replacing stent. A diuretic T1/2 showed the improvement of differential renal function without urinary obstruction in all patients. CONCLUSION: Laparoscopic Yang-Monti ureteral reconstruction is safe and feasible in children with an excellent outcome.

Beta-adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca2+ channel.

As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.

Beta-nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle.

Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that beta-nicotinamide adenine dinucleotide (beta-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of beta-NAD mimics the endogenous neurotransmitter better than ATP. Levels of beta-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of beta-NAD depends on the frequency of nerve stimulation. beta-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or omega-conotoxin GVIA. beta-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous beta-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to beta-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. beta-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.

[Electrophysiology of two human hyperpolarization activated cyclic nucleotide-gated potassium channels expressed in HEK293 cells].

OBJECTIVE: To express the human HCN2 and HCN4 genes in HEK293 cells and investigate the electrophysiology of the expressed channel protein. METHODS: cDNA encoding human HCN2 or HCN4 gene was ligated into a shuttle vector pAdTrack-CMV. Homologous recombination was performed in Escherichia coli of the line BJ5183. Human embryonic kidney cells of the line 293 (HEK293 cells) were cultured and transfected with the positive recombinant adenovirus plasmid. Then the HEK293 cells were infected by AdhHCN2 or AdhHCN4 and the whole cell hyperpolarization-activated currents were recorded in HEK293 cells transfected with hHCN2 and hHCN4. RESULTS: If-like currents could be found in the HEK293 cells transfected with hHCN2 and hHCN4. The channels were activated by hyperpolarized potentials. Boltzmann equation showed that the half-activation voltage of the hHCN2 and hHCN4 channels were -114.8 mV +/- 3.3 mV and -125.9 mV +/- 2.9 mV respectively (P = 0.024). The reversal slope factors of the hHCN2 and hHCN4 channels were 11.1 mV +/- 1.2 mV and 13.7 mV +/- 1.3 mV respectively (P = 0.22). The activation kinetics was faster in hHCN2 than in hHCN4, with the activation constants at -110 mV being 0.99 s +/- 0.21 s and 8.47 s +/- 2.85 s respectively. The relative permeation ratio for sodium and potassium were 0.40 and 0.34 respectively in these two channels. Caesium chloride of the concentration of 2 mmol/L prominently inhibited both currents. CONCLUSION: The target genes hHCN2 and hHCN4 are successfully expressed in HEK293 cells, and the expressed functional channels have profoundly different activation kinetics.

Transgenic expression of a putative calcium transporter affects the time of Arabidopsis flowering.

PPF1 is a gibberellin-induced, vegetative growth-specific gene, first isolated from short-day (SD)-grown G2 pea plants. In the current work, we found that transgenic Arabidopsis plants overexpressing the PPF1 gene (PPF1 (+)) flowered much later and had a significantly longer lifespan compared to control plants, whereas suppression of this gene (PPF1 (-)) resulted in a very rapid reproductive cycle. Western blotting analyses of PPF1 (+) and (-) plant lines revealed a positive correlation between the amount of antibody-reactive protein and the time of flowering. Green fluorescent protein (GFP) co-expression assays showed that the PPF1 protein is likely localized in chloroplast membranes. Transgenic expression of PPF1 affected the calcium storage capacities since chloroplasts isolated from PPF1 (+) plants contained high Ca2+ levels while chloroplasts of PPF1 (-) plants contained very low amounts of calcium ion. Using Novikoff human hepatoma cells, we demonstrated that expression of PPF1 leads to a significant inward calcium ion current that was absent in untransformed cells. We conclude that, as a putative calcium ion carrier, PPF1 affects the flowering time of higher plants by modulating Ca2+ storage capacity within chloroplasts.