Localization by immunofluorescence of a gastrin-like substance in the brain of the rainbow trout,Salmo gairdneri

Summary With the aid of an indirect immunofluorescence technique neurones containing a gastrin-like substance were identified in the brain of Salmo gairdneri. The perikarya of these neurones appear to be located along the periventricular part of the nucleus lateralis tuberis between the hypophysial stalk and the most rostral tip of the saccus vasculosus. The fibres of these perikarya run rostrally toward the hypophysis, where they can be followed in the protrusions of the neurohypophysis into the proximal pars distalis. Here the bundle of immunoreactive fibres divides into numerous smaller bundles and into single fibres. Immunohistochemical specificity tests have shown this immunoreactive substance to belong to the gastrin group, sharing an antigenic determinant with cholecystokinin (CCK) and pentagastrin (common aminoacid sequence Trp-Met-Asp-Phe). A possible function of these gastrin (or CCK)-containing neurones in the rainbow trout is discussed.     

肝泡状棘球蚴病不同手术疗效及预后的分析研究

目的:比较肝泡状棘球蚴病(HAE)不同手术方式的疗效,并分析影响HAE手术患者预后的相关因素。方法:回顾性分析2003年9月-2015年2月期间在我院进行手术治疗的HAE病人的诊疗记录。根据手术方式的不同,将病人分为非移植性根治性切除组(A组)、术中病灶绝大部分切除(90%以上)组(B组)、术中不能90%以上切除或仅引流组(C组)、肝移植组(D组)。结合随访资料,评价四组的疗效,并分析影响患者预后的相关因素。结果:A组死亡率低于其他三组,差异具有统计学意义(P0.001)。生存曲线结果显示,A组预后生存状况优于其他三组,差异具有统计学意义(P=0.001)。多因素分析结果表明,非移植性根治性切除、术中出血量是影响患者生存的独立危险因素(均P0.05)。结论:在早期发现早期诊断的前提下,对HAE病人行非移植性根治性切除术治疗效果最好,且非移植性根治性切除是患者预后的独立危险因素。     

Paeoniflorin diminishes ConA-induced IL-8 production in primary human hepatic sinusoidal endothelial cells in the involvement of ERK1/2 and Akt phosphorylation

Liver diseases are closely associated with elevated levels of interleukin-8 (IL-8), suggesting the ability to inhibit IL-8 production could enhance the treatment of liver diseases. Paeoniflorin is a major active constituent of dried Paeoniae Radix Alba root (Baishao in Chinese) which is widely used in China to treat liver diseases. We examined the effects and underlying mechanisms of paeoniflorin on IL-8 production in primary human hepatic sinusoidal endothelial cells (HHSECs). Concanavalin A (ConA) at 20 μg/mL produced a 5.2-fold increase in IL-8 mRNA by 8 h, and a 14.2-fold rise in IL-8 levels by 16 h. Inhibition of MEK (ERK kinase) and extracellular signal-regulated kinase (ERK) by PD98059 and U0126, or inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked both ConA-induced IL-8 mRNA expression and IL-8 secretion. Paeoniflorin reduced ConA-induced IL-8 mRNA expression and IL-8 release by 57.9% and 52.8%, respectively, and also decreased ConA-stimulated phosphorylation of ERK1/2 and Akt, suggesting paeoniflorin inhibits IL-8 expression and release by inhibiting the ERK1/2 and Akt pathways. Combining paeoniflorin with U0126 or LY294002 at low doses showed supra-additive inhibition of not only phospho-ERK1/2 and phospho-Akt by 46.4% and 35.0%, but also IL-8 release by 42.4% and 36.1% and IL-8 mRNA expression by 43.5% and 31.8%, respectively. In conclusion, paeoniflorin most likely contributes to the therapy for liver disease by exerting anti-inflammatory effects on HHSECs through blocking IL-8 secretion via downregulation of ERK1/2 and Akt phosphorylation.     

Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.     

血清铜蓝蛋白联合透明质酸检测在肝纤维化患者诊断中的应用

目的探讨血清铜蓝蛋白(CP)联合透明质酸(HA)检测在肝纤维化患者诊断中的应用前景。方法选取2015年1月至2018年12月本院收治的疑似肝纤维化患者226例为研究对象,对患者的CP、HA水平进行联合检测,根据各项指标检测水平对患者做出诊断,并与金标准诊断方法肝穿刺活检结果进行对比。对CP、HA单独检测和联合检测的诊断结果进行统计对比,并对各组检查方法的灵敏度、特异度、假阳性率、假阴性率、准确度等进行统计对比,比较不同检查方法在不同肝纤维化分级患者中的诊断正确率。结果 226例患者中金标准诊断201例(88.94%)为肝纤维化,CP单独检测检出154例(68.14%),HA单独检测检出143例(63.27%),CP+HA联合检测检出195例(86.28%)。经Kappa一致性检验分析CP+HA联合检测与金标准结果有高度一致性,CP、HA单独检测与金标准结果为中等一致性。CP+HA联合检测的灵敏度、特异度、准确度均高于CP、HA单独检测,方法间比较差异有统计学意义(χ~2=11.602、55.959、78.047,P=0.003、0.039、0.002)。在不同分级肝纤维化患者中,CP+HA联合检测在Ⅰ级、Ⅱ级纤维化检出率高于CP、HA单独检测,方法间比较差异有统计学意义(χ~2=9.432、8.324,P=0.007、0.010)。结论 CP联合HA检测在肝纤维化患者诊断中应用情况良好,有较高的诊断正确率,且对症状程度较轻的肝纤维化患者有较高的检出率,可作为肝纤维化诊断检查的主要手段加以推广应用。     

Characteristics of Wine Produced by Mushroom Fermentation

Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

不同二步酶灌注法分离大鼠肝星状细胞的比较研究

目的:在现有二步酶灌注法分离大鼠肝星状细胞(hepatic stellate cells,HSC)的基础上,探索更加高效的分离HSC方法。方法:分别采用链酶蛋白酶+胶原酶循环灌注、链酶蛋白酶非循环灌注+胶原酶循环灌注以及胶原酶单独循环灌注法分离大鼠HSC,比较三种方法的细胞获得率、活性和纯度差异。应用0.4%台盼蓝染色判断活性,结蛋白(desmin)、波形蛋白(vimentin)细胞免疫荧光方法鉴定纯度。结果:链酶蛋白酶非循环灌注+胶原酶循环灌注法细胞获得率高于另两种方法,细胞活力高于链酶蛋白酶循环灌注+胶原酶循环灌注法,三组得到的细胞纯度均高于90%且无显著差异。结论:在三种二步酶灌注方法中,链酶蛋白酶非循环灌注+胶原酶循环灌注法能显著提高HSC获得率,且对细胞活力影响小,不降低细胞纯度,是一种高效的分离方法,有利于HSC相关肝脏疾病的生物学研究。     

Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Drug substance (DS) color is an important quality attribute for release, stability and comparability studies of biologics. With the increase of DS concentrations and biologics pipelines made in chemically defined media, atypical DS color other than colorless or pale yellow has been recently reported in the biopharmaceutical industry. We recently observed a brown DS color in manufacturing. Although analytical characterization data indicated that the brown color DS had no major quality issue, it is necessary to find the root cause and reduce DS color to ease placebo design for clinical use. It was demonstrated that the brown color was caused by the chemically defined basal medium containing high levels of iron and vitamin B12 (VB12) regardless of cell lines. Iron caused tryptophan oxidation in the protein to form N-formylkynurenine and kynurenine products, which likely contributed to a yellow DS color. A pink DS color was caused by the residual VB12 bound to DS. The brown color was the result of the combinatory effect of yellow and pink colors. Finally a modified basal medium was developed to produce a pale yellow DS in manufacturing.     

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice.Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.