STAT3 couples with 14-3-3σ to regulate BCR signaling,B-cell differentiation,and IgE production

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Pde4d和Alox5ap的mRNA水平与高血压性脑卒中和高血压病相关性的动物实验研究

目的使用与人类高血压性脑卒中近似的模型大鼠——易卒中型肾血管性高血压大鼠(stroke-prone renovascular hypertensive rats,RHRSP),在动物水平评价脑卒中易感基因Pde4d和Alox5ap mRNA水平的变化规律与高血压性脑卒中和高血压病的相关性。方法建立血压正常组,梯度高血压Ⅰ、Ⅱ和Ⅲ组(其收缩压分别为140～159mmHg、160～179mmHg和180～199mmHg)和自发卒中组,分别提取大鼠外周血的总RNA,逆转录后进行荧光实时定量PCR。结果自发卒中组Pde4d和Alox5ap的mRNA表达水平明显高于其他组。Pde4d在梯度高血压Ⅰ组的表达量稍高于血压正常组、梯度高血压Ⅱ、Ⅲ组;Alox5ap在血压正常组和梯度高血压组的表达量差异无统计学意义。结论动物实验表明Pde4d与Alox5ap基因过表达均与高血压性脑卒中存在相关性,Pde4d在梯度高血压Ⅰ组表达稍高可能与大鼠机体应激相关;两基因与高血压病本身无显著相关性,是脑卒中的独立危险因素;两基因的过表达可能通过参与动脉壁的炎症反应而介导脑卒中的发生。     

Novel Brugada syndrome‐causing mutation in ion‐conducting pore of cardiac Na+ channel does not affect ion selectivity properties

Aim: Brugada syndrome is an inherited cardiac disease with an increased risk of sudden cardiac death. Thus far Brugada syndrome has been linked only to mutations in SCN5A, the gene encoding the α‐subunit of cardiac Na⁺ channel. In this study, a novel SCN5A gene mutation (D1714G) is reported, which has been found in a 57‐year‐old male patient. Since the mutation is located in a segment of the ion‐conducting pore of the cardiac Na⁺ channel, which putatively determines ion selectivity, it may affect ion selectivity properties. Methods: HEK‐293 cells were transfected with wild‐type (WT) or D1714G α‐subunit and β‐subunit cDNA. Whole‐cell configuration of the patch‐clamp technique was used to study biophysical properties at room temperature (21 °C) and physiological temperature (36 °C). This study represents the first measurements of human Na⁺ channel kinetics at 36 °C. Ion selectivity, current density, and gating properties of WT and D1714G channel were studied. Results: D1714G channel yielded nearly 80% reduction of Na⁺ current density at 21 and 36 °C. At both temperatures, no significant changes were observed in V_1/2 values and slope factors for voltage‐dependent activation and inactivation. At 36 °C, but not at 21 °C, D1714G channel exhibited more slow inactivation compared with WT channel. Ion selectivity properties were not affected by the mutation at both temperatures, as assessed by either current or permeability ratio. Conclusion: This study shows no changes in ion selectivity properties of D1714G channel. However, the profoundly decreased current density associated with the D1714G mutation may explain the Brugada syndrome phenotype in our patient.     

B族链球菌C5a肽酶蛋白表位的克隆、表达和免疫学分析

目的应用生物信息学方法预测B族链球菌C5a肽酶蛋白表位，结合基因工程手段进行表位重组、表达和免疫原性分析。方法用预测程序ProPred和ANTIGENIC预测B族链球菌C5a肽酶蛋白的表位，应用PCR技术扩增出编码该表位基因片段，克隆PCR产物构建重组质粒，测序验证。在大肠杆菌BL21（DE3）中诱导表达融合蛋白。表达的蛋白经质谱分析和Western blot鉴定。纯化该融合蛋白并免疫C57／BL小鼠，萃取GBS表面蛋白，双向琼脂扩散试验检测抗体水平。结果在SCPB中预测到1个既具有MHC结合肽特性又具有B细胞表位特征的肽段。重组和表达了这一肽段，质谱得出与SCPB蛋白的相似性分数为79，Western-blot证实能与抗SCPB的抗体反应，纯化后融合蛋白纯度〉90％。动物实验证实融合蛋白能产生特异性的抗GBS抗体。结论重组表位具有一定免疫原性。为相关蛋白的毒力机制研究和亚单位疫苗等方面的研究打下了良好的基础。     

Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics

We generated a high-growth H5N1/PR8 virus by plasmid-based reverse genetics. The virulence associated multiple basic amino acids of the HA gene were removed, and the resulting virus is attenuated for chickens and chicken eggs. A formalin-inactivated oil-emulsion vaccine was prepared from this virus. When SPF chickens were inoculated with 0.3 ml of the vaccine, the hemagglutinin-inhibition (HI) antibody became detectable at 1 week post-vaccination (p.v.) and reached a peak of 10log2 at 6 weeks p.v. then slowly declined to 4log2 at 43 weeks p.v. Challenge studies performed at 2, 3 and 43 weeks p.v. indicated that all of the chickens were completely protected from disease signs and death. Ducks and geese were completely protected from highly pathogenic H5N1 virus challenge 3 weeks p.v. The duration of protective immunity in ducks and geese was investigated by detecting the HI antibody of the field vaccinated birds, and the results indicated that 3 doses of the vaccine inoculation in geese could induce a 34 weeks protection, while 2 doses induced more than 52 weeks protection in ducks. We first reported that an oil-emulsion inactivated vaccine derived from a high-growth H5N1 vaccine induced approximately 10 months of protective immunity in chickens and demonstrated that the oil-emulsion inactivated avian influenza vaccine is immunogenic for geese and ducks. These results provide useful information for the application of vaccines to the control of H5N1 avian influenza in poultry, including chickens and domestic waterfowl.     

Effects of C5a and FMLP on Interleukin-8 Production and Proliferation of Human Umbilical Vein Endothelial Cells

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1, TNF, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1 and TNF significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1, TNF, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a ³H-thymidine incorporation assay. In contrast with IL-1 and TNF, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.     

Behavioral characteristics of rat lines selected for differential hypothermic responses to cholinergic or serotonergic agonists

The present review will describe the formation of two pharmacologically selected lines of rats, their behavioral phenotypes, their responses to select drugs, their possible neurochemical correlates, and their use to detect the therapeutic potential of antidepressant drugs. The Flinders Line rats were established at Flinders University in Australia by selectively breeding for differential responses to an anticholinesterase agent from outbred Sprague-Dawley (SD) rats; the Flinders Sensitive Line (FSL) rats were more sensitive to the hypothermic and behavioral suppressing effects of this agent than the Flinders Resistant Line (FRL) rats. The 8-OH-DPAT line rats were established at the University of North Carolina at Chapel Hill by selectively breeding for differential hypothermic responses to the 5-HT_1A receptor agonist, 8-OH-DPAT; the high DPAT sensitive (HDS) line rats were more sensitive to the hypothermic effects of 8-OH-DPAT than the low DPAT sensitive (LDS) line rats. Studies of these two pairs of lines have indicated that the FSL and HDS rats are both more susceptible to stress-induced behavioral disturbances. Their usefulness in detecting potential antidepressant drugs and the relationship between mood disorders and drug abuse will be discussed.     

The molecular basis for inherited bullous diseases

In the past 5 years enormous progress have been made in our understanding of the molecular basis for a number of inherited skin diseases characterized by easy blistering of the skin and the mucous membranes after minor physical trauma. This increased fragility of the skin or its appendages is due to molecular defects in genes coding for different intra- and extracellular structural proteins which are responsible for mechanical strength at their sites of expression. These diseases encompass the group of epidermolysis bullosa and disorders of cornification such as bullous forms of ichthyosis, palmoplantar keratoderma, and pachyonychia congenita. On the basis of clinical, morphological, and ultrastructural observations the epidermolysis bullosa group has been divided into three major categories. In epidermolysis bullosa simplex blister formation appears within the basal cell layer of the epidermis, and many mutations have been found in the genes of keratin 5 and 14 which are both expressed in basal keratinocytes. Epidermolytic hyperkeratosis leads to an epidermal separation in the suprabasal cell layers. In these patients numerous point mutations have now been described in the suprabasally expressed genes of keratin 1 and 10. In ichthyosis bullosa of Siemens blisters occur in the more upper suprabasal epidermis coincidental with the expression of keratin 2e, and mutations have been detected in the corresponding gene. In epidermolytic palmoplantar hyperkeratosis the suprabasal epidermal splitting is restricted to palms and soles of the patient. In keratin 9, which reveals such an exclusive expression pattern, molecular defects have indeed been recognized. Most recently in two different clinical subtypes of pachyonychia congenita, which is characterized by defective nails and focal palmoplantar hyperkeratosis, point mutations have been found in the genes coding for keratins 6, 16, and 17. In junctional epidermolysis bullosa the separation takes place within the dermal-epidermal basement membrane at the level of the lamina lucida, and mutations have been found in three genes coding for different laminin chains, in the ₄ gene of ₆₄ integrin, and in the gene of collagen XVII. In dystrophic epidermolysis bullosa the tissue separation occurs beneath the basement membrane within the papillary dermis at the level of the anchoring fibrils, and several mutations have been identified in the collagen VII gene. The rapid unraveling of molecular defects in these disabling or even lethal inherited skin diseases makes possible a more precise and earlier prenatal diagnosis, creates new options for suitable therapeutic regimens, and even offers the hope of curing these diseases by means of somatic cell gene therapy.Abbreviations BM Basement membrane - BPAg Bullous pemphigoid antigen - DEB Dystrophic epidermolysis bullosa - EB Epidermolysis bullosa - EBS Epidermolysis bullosa simplex - EHK Epidermolytic hyperkeratosis - EPPK Epidermolytic palmoplantar keratoderma - IBS Ichthyosis bullosa of Siemens - JEB Junctional epidermolysis bullosa - KIF Keratin intermediate filaments - NC Noncollagenous domain - NEPPK Nonepidermolytic palmoplantar keratoderma - PC Pachyonychia congenita     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.