Molecular characterization of Pacific white shrimp (Litopenaeus vannamei) sodium bicarbonate cotransporter (NBC) and its role in response to pH stress.

The sodium bicarbonate cotransporter (NBC) is an integral membrane ion transporter that can transport HCO3- (or a related species, such as CO32-) across the plasma membrane. Previous researches revealed that NBC might play an important role in the regulation of intracellular pH in vertebrates. In the present study, an NBC cDNA was identified from Pacific white shrimp (Litopenaeus vannamei) and designated as Lv-NBC. The full-length Lv-NBC cDNA is 4479 bp in size, containing a 5'-untranslated region (UTR) of 59 bp, a 3'-UTR of 835 bp and an open reading frame (ORF) of 3585 bp that encodes a protein of 1194 amino acids with a deduced molecular weight of 134.34 kDa. The Lv-NBC protein contains two functional domains (Band_3_cyto and HCO3_cotransp) and twelve transmembrane (TM) domains. Expression of the Lv-NBC mRNA was ubiquitously detected in all selected tissues, with the highest level in the gill. By in situ hybridization (ISH) with Digoxigenin-labeled probe, the Lv-NBC positive cells were shown mainly located in the secondary gill filaments. After low or high pH challenge, the transcript levels of Lv-NBC in the gill were found to be up-regulated. After knockdown of the Lv-NBC level by siRNA, the mortality of shrimp significantly increased under pH stress. Our study, as a whole, may provide evidences for the role of NBC in shrimp responding to pH stress, and give a new insight of the acid/base homeostasis mechanism in crustaceans.

Goldfish Leptin-AI and Leptin-AII: Function and Central Mechanism in Feeding Control.

In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model.

Engineering of an enhanced synthetic Notch receptor by reducing ligand-independent activation.

Notch signaling is highly conserved in most animals and plays critical roles during neurogenesis as well as embryonic development. Synthetic Notch-based systems, modeled from Notch receptors, have been developed to sense and respond to a specific extracellular signal. Recent advancement of synNotch has shown promise for future use in cellular engineering to treat cancers. However, synNotch from Morsut et al. (2016) has a high level of ligand-independent activation, which limits its application. Here we show that adding an intracellular hydrophobic sequence (QHGQLWF, named as RAM7) present in native Notch, significantly reduced ligand-independent activation. Our enhanced synthetic Notch receptor (esNotch) demonstrates up to a 14.6-fold reduction in ligand-independent activation, without affecting its antigen-induced activation efficiency. Our work improves a previously reported transmembrane receptor and provides a powerful tool to develop better transmembrane signaling transduction modules for further advancement of eukaryotic synthetic biology.

[Correlation of toll-like receptor 3 and tumor necrosis factor-alpha with idiopathic fetal growth restriction.].

OBJECTIVE: To investigate the expression and the significance of toll-like receptor 3 (TLR-3) in placenta, tumor necrosis factor-alpha(TNF-alpha) in maternal and cord blood of idiopathic fetal growth restriction (IFGR), and their correlation with the pathogenesis of symmetric and asymmetric IFGR. METHODS: From April 2008 to April 2009, 42 primiparae of singleton pregnancy and their IFGR babies, who delivered at term through cesarean section, in the Third Affiliated Hospital of Zhengzhou University were enrolled. All subjectects were divided into symmetric IFGR group (n = 20) and asymmetric IFGR group (n = 22). Another 42 non-IFGR pairs were randomly selected as the control group. The polink-2 plus polymerized horseradish peroxidase (HRP) immunohistochemical method and the enzyme linked immunosorbent assay (ELISA) were applied to detect TLR-3 and TNF-alpha levels. RESULTS: (1) The expression of TLR-3 protein were observed in all maternal placenta of the three groups. TLR-3 essentially expressed in syncytiotrophoblasts and hofbouer cells in the symmetric IFGR and control group, but expressed mostly in hofbouer cells and less in syncytiotrophoblasts in the asymmetric IFGR group. (2) The expression of TLR-3 in the syncytiotrophoblasts of the symmetric and asymmetric IFGR group was significantly lower than in the control group (111 +/- 14 and 118 +/- 11 vs. 156 +/- 9, P < 0.01). The number of TLR-3 positive in Hofbourer cell in the symmetric IFGR group was lower than the control group (8.9 +/- 2.8 vs 17.5 +/- 2.8, P < 0.01), but the number in the asymmetric IFGR group was higher (23.8 +/- 3.7) compared with the control group (P < 0.01). (3) The TNF-alpha levels in the maternal and cord blood of the symmetric and the asymmetric group were higher than that of the control group [maternal: (90 +/- 10) microg/L and (86 +/- 11) microg/L vs. (73 +/- 9) microg/L; cord blood: (92 +/- 12) microg/L and (96 +/- 8) microg/L vs. (79 +/- 9) microg/L; P < 0.01]. (4) Neither symmetric nor the asymmetric IFGR group showed any correlations between the maternal and cord blood levels of TNF-alpha (P > 0.05). (5) Significant correlation was found between the TNF-alpha level of the cord blood and TLR-3 expression in the placenta in both the symmetric and asymmetric IFGR group (P < 0.05), but no relationship was found between the maternal blood TNF-alpha level and TLR-3 expression in the placenta (P > 0.05). CONCLUSIONS: The variantions of TLR-3 expression in placenta and the increased expression of TNF-alpha in cord blood are associated with the genesis IFGR. The reduced expression of TLR-3 may related to symmetric IFGR, while the increased TLR-3 level in hofbouer cells may lead to asymmetric IFGR.

[An experimental study of expression of angiotension converting enzyme 2 in myocardium and effect of telmisartan treatment in pressure-overloaded rats].

OBJECTIVE: To study the effect of hypertension and telmisartan treatment on the protein and gene expression of cardiac angiotensin-converting enzyme 2 (ACE2) in pressure-overloaded rats. METHODS: Coarctation of suprarenal abdominal aorta was reproduced in 8 week-old male Sprague-Dawley (SD) rats and then randomized into 4 groups, including a sham group (n=15), a suprarenal aortic coarctation group (model group, n=12), a suprarenal aortic coarctation with low-dose Telmisartan treatment group (low-dose group, 2 mgxkg(-1)xd(-1), n=11) and a suprarenal aortic coarctation with high-dose Telmisartan treatment group(high-dose group, 10 mgxkg(-1)xd(-1), n=13). Telmisartan or equivalent amount of normal saline was gavaged 24 hours before the operation and once every day afterwards for 3 weeks. At the end of 3 weeks, the concentrations of angiotensin (AngII) in plasma and myocardium were measured by radioimmunoassay. Changes in both protein quantity and gene expressions of both ACE2 and ACE were determined by Western blotting analysis and reverse transcription-polymerase chain reaction (RT-PCR) technique, respectively. RESULTS: Suprarenal abdominal aortic coarctation induced a significant increase in the plasma and myocardium AngII concentration [plasma: (495.1+/-55.6) ng/L vs. (269.2+/-39.5)ng/L, myocardium: (103.6+/-23.7) ng/g vs. (49.5+/-13.5) ng/g, both P<0.01] and expressions of gene and protein of ACE (P<0.01) and ACE2 (P<0.05). Telmisartan further increased the concentration of AngII in plasma and myocardium in a dose-dependent manner [plasma: (702.2+/-40.6) ng/L vs. (612.6+/-35.5) ng/L, myocardium (211.5+/-21.5) ng/g vs. (189.6+/-43.6) ng/g, both P<0.05], and induced a dose-dependent increase in both protein and gene expression of ACE2 (protein 1.16+/-0.06 vs. 0.79+/-0.04, gene 0.54+/-0.08 vs. 0.41+/-0.04, both P<0.05). Expression of ACE2 protein in low-dose and high-dose groups was increased by 1.0 and 1.58 folds respectively, and gene was increased by 1.3 and 1.6 folds (all P<0.05). The expression of ACE protein and gene in model group increased significantly (protein: 2.10+/-1.07 vs. 1.02+/-0.05, gene: 1.93+/-0.09 vs. 0.26+/-0.09, both P<0.01). Telmisartan had no significant effect on ACE gene and protein expressions (both P>0.05). CONCLUSION: Suprarenal abdominal aortic coarctation induced a significant increases of ACE and ACE2 gene and protein expressions. Telmisartan induces a dose-dependent increases of cardiac ACE2 gene and protein expression,which may be the mechanism of its therapeutic effects.

A risk-predictive score for cardiogenic shock after acute myocardial infarction in Chinese patients.

BACKGROUND: Cardiogenic shock after acute myocardial infarction (AMI) remains a poor prognosis. Although numerous studies discussed the predictors of cardiogenic shock complicating AMI, the data in Chinese patients is still absent. The goal of this study is to develop a risk-predictive score for cardiogenic shock after AMI, among Chinese patients, so as to guide clinicians to prevent cardiogenic shock. METHODS: Patients with ST-segment elevated AMI were provided by two Chinese hospitals from 1994 to 2004. Baseline characteristics of each case were documented. Multivariable logistic regression modeling techniques were used to develop a model to predict the occurrence of cardiogenic shock within 72 h after admission. On the basis of the coefficients in the model, a risk score was developed for the probability of cardiogenic shock. To test its viability, another population, which was consistent with the original population, confirmed the scoring. RESULTS: Among 2,077 patients, 184 cases developed cardiogenic shock within 72 h. Age, gender, BMI, killip class, MI location, multivessel disease, previous MI, family history of CAD, and thrombolytic therapy were strong predictors for shock after AMI. A risk-predictive score for shock was developed. It predicted cardiogenic shock accurately in another Chinese population. CONCLUSIONS: A predictive model is developed in Chinese patients with AMI for the first time. It is based on some simple parameters, which can be easily obtained by clinicians. The risk score derived from the model can predict cardiogenic shock accurately.

Evaluation of 2-stage Treatment for Cervical Dorsal Rami Entrapment Syndrome: A Randomized, Controlled Trial.

OBJECTIVES: To evaluate the effectiveness of a 2-stage nonoperative treatment for patients with cervical dorsal rami entrapment syndrome. MATERIALS AND METHODS: The study included 66 patients diagnosed with cervical dorsal rami entrapment syndrome randomized to an experimental group (n=33) and control group (n=33). The experimental group was treated with additional diagnostic block if regular 2 weeks medication was not effective. The control group only received nonsteroidal anti-inflammatory drugs for 2 weeks. A visual analog scale (VAS) and pain treatment satisfaction scale (PTSS) were used to assess pain. Muscle power in the upper limbs was also assessed. The registration number of this study is ChiCTR-IIR-15007565. RESULTS: The VAS scores of the experimental group were significantly lower at 2, 4, and 6 months after treatment compared with baseline and the VAS scores of the control group (all P<0.001). The PTSS scores of the experimental group were significantly higher at 2, 4, and 6 months after treatment compared with baseline and the PTSS scores of the control group (all P<0.001). Maximal muscle power after treatment was significantly greater in the experimental group compared with the control group for shoulder abduction (P<0.001), thumb pinch force (P=0.001), and grasp (P<0.001). CONCLUSIONS: The results suggest that the 2-stage treatment is effective for patients with cervical dorsal rami entrapment syndrome.

[Study on up-regulation of the expression of angiotensin-converting enzyme-2 in human umbilical vein endothelial cells by telmisartan].

OBJECTIVE: To investigate the effect of telmisartan on the protein and gene expression of angiotensin-converting enzyme-2 (ACE2) in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with various concentrations of telmisartan (10(-7), 10(-6) and 10(-5) mol/L) for 24 hours. In a time-control experiment, HUVECs were treated with telmisartan at the final concentration of 10(-6) mol/L for 6, 12 and 24 hours, respectively. In another experiment, HUVECs were treated with PD123319 (10(-6) mol/L) only or combined with same final concentration of telmisartan for 12 hours, respectively. Changes in both protein and gene expression of ACE2 were determined with Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) technique, respectively. RESULTS: Telmisartan induced a concentration and time dependent increase in both protein and gene expression of ACE2 (P<0.05 or P<0.01). Compared with control group, treatment of HUVECs with telmisartan at the concentration of 10(-7), 10(-6) and 10(-5) mol/L stimulated 1.5-, 2.7- and 4.6-fold increase in the ACE2 protein expression, as well as 1.2-, 2.3- and 4.5-fold increase in its gene expression, respectively. After treatment of HUVECs with telmisartan for 6, 12, and 24 hours at the concentration of 10(-6) mol/L, the ACE2 protein expression increased 1.6-, 2.7- and 4.2-fold, and its gene expression increased 1.3-, 2.3- and 4.0-fold, respectively. Compared with control and telmisartan groups, PD123319 had no effect on both protein and gene expression of ACE2 (P>0.05). CONCLUSION: Telmisartan up-regulates the protein and gene expression of ACE2 in HUVECs in a concentration and time dependent manner. This effect may be mediated via its specific pathway.

[Angiotensin-(1-7) modulates fibrinolytic imbalance induced by oxidized low-density lipoprotein in cultured human umbilical vein endothelial cells].

OBJECTIVE: To study the effect of angiotensin-(1-7)[Ang-(1-7)]on fibrinolytic imbalance induced by oxidized low- density lipoprotein (ox-LDL) in cultured human umbilical vein endothelial cells (HUVECs). METHOD: Cultured HUVECs were incubated for 24 h in the presence of Ang-(1-7), ox-LDL and A-779 at different concentrations either separately or in combination. The final concentrations of Ang-(1-7) were 10, 100 and 1,000 nmol/L, and those of ox-LDL were 25, 50 and 100 mg protein/L. The final concentration of A-779, an Ang-(1-7) receptor antagonist, was 100 nmol/L. Tissue plasminogen activator (t-PA) and its inhibitor-1(PAI-1) antigen in the medium were determined by enzyme-linked immunosorbent assay (ELISA) and their mRNA levels by reverse transcriptional (RT) PCR. RESULTS: Ox-LDL (25-100 mg protein/L) dose-dependently increased PAI-1 release and up-regulated PAI-1 gene transcription, but decreased t-PA release and down-regulated t-PA gene transcription (P>0.001-0.05). Ang-(1-7) (100-1,000 nmol/L) dose-dependently decreased PAI-1 release and PAI-1 gene transcription (P>0.01-0.05) but had no effect on t-PA release and gene transcription. In the presence of ox-LDL, Ang-(1-7) lowered the increased levels of PAI-1 and PAI-1 mRNA, and elevated the levels of decreased t-PA and t-PA mRNA in the cells (P>0.01-0.05). The effects of Ang-(1-7) could be blocked by A-779. CONCLUSION: Angiotensin-(1-7) effectively modulates the fibrinolytic imbalance induced by ox-LDL via its specific receptor.

Chronic administration of angiotensin-(1-7) attenuates pressure-overload left ventricular hypertrophy and fibrosis in rats.

OBJECTIVE: To test the hypothesis that chronic administration of angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac hypertrophy in rats in vivo. METHODS: Coarctation of the suprarenal abdominal aorta was performed in 41 8-week-old male Sprague Dawley rats. Twenty-four hours after the operation, osmotic minipumps were surgically implanted subcutaneously in the rats, which were randomly divided into 3 groups, including a sham-operation group (n=15) receiving infusion with normal saline, a suprarenal aortic coarctation group (n=12), and a suprarenal aortic coarctation group (n=14) with Ang-(1-7) treatment at the dose of 25 mug x kg(-1) x h(-1). Four weeks later, the systolic and diastolic blood pressures were measured and the left ventricular mass index (LVMI, mg/g) was calculated from the ratio of left ventricular weight to body weight. The concentrations of Ang II in the plasma and myocardium were measured by radioimmunoassay, and myocardial interstitial collagen volume fraction (ICVF) was determined by quantitative morphometry of the sections with Picrosirius red staining using an automated image analyzer. RESULTS: Suprarenal abdominal aortic coarctation induced a significant increase in carotid artery systolic and diastolic blood pressure, heart weight, LVMI, ICVF, and the concentration of Ang II in the myocardium (P<0.01). Chronic administration of Ang-(1-7) attenuated the increase in the heart weight, LVMI, ICVF and left ventricular diastolic end pressure (LVEDP) caused by suprarenal abdominal aortic coarctation (P<0.05). Ang-(1-7) also increased the formerly decreased maximum left ventricular pressure reduction rate (-dP/dt(max)) (P<0.05), but had no effect on blood pressure and the concentration of Ang II in the myocardium. No difference was noted in plasma concentration of Ang II between the 3 groups. CONCLUSIONS: Ang-(1-7) attenuates cardiac hypertrophy and fibrosis and preserved the impaired left ventricular function induced by left ventricular pressure-overload in rats. These effects are not associated with the changes in the concentrations of Ang II in the left ventricular myocardium and plasma.