Formation of complexes between avidin and β-adrenergic receptors using biotinyl-alprenolol derivatives

The goal of this study was to synthesize biotinylated derivatives of alprenolol, a β-adrenergic antagonist, and to determine whether these ligands could bind simultaneously to both avidin (a biotin-binding protein) and to the β-adrenergic receptor. Such ligands would be useful for β-adrenergic receptor localization and purification, since avidin can be covalently labelled with fluorescent or electron-dense markers or can be linked to solid supports for affinity chromatography. Three biotinyl derivatives of alprenolol were synthesized and characterized. Each derivative bound to avidin and also possesed high affinity for the duck erythrocyte β-adrenergic receptor. Two of the compounds, biotinyl-caproyl-cysteaminyl-alprenolol (BCCA) and biotinyl-dodecanoyl-cysteaminyl-alprenolol (BDCA) had the same affinities for the duck erythrocyte β-adrenergic receptor (membrane-bound or digitonin-solubilized) in the absence and presence of avidin. This indicated that high affinity complexes could be formed between the β-adrenergic receptor and avidin using these bifunctional biotinyl-alprenolol ligands. In contrast, biotinyl-cysteaminyl-alprenolol (BCA), in which the distance between the biotin and alprenolol moieties was shorter, had greatly reduced affinity for the duck erythrocyte β-adrenergic receptor in the presence of avidin. Additional studies showed that BDCA, avidin-BDCA, and ferritin-avidin-BDCA were equally potent in inhibiting the isoproterenol stimulation of cAMP accumulation in intact HeLa cells. The data reported in this paper demonstrate the importance of an appropriate spacer sequence to allow correct apposition of the receptor and avidin molecules, and suggest that BDCA may be a useful probe for β-adrenergic receptor localization and purification.     

北京鸭血清HDL主要载脂蛋白的高效液相层析分离及其理化性质

用高效液相层析(HPLC)对北京鸭血清高密度脂蛋白(HDL)的所有载脂蛋白(apo)分离纯化,得五个主要apo峰,经SDS-PAGE鉴定,均为单一带;并做了IEF、糖基含量分析;对其中含量高的四个apo峰样进行了氨基酸组成测定和N-端部分氨基酸序列分析;从所做理化性质的研究,发现北京鸭血清HDL中的主要apo为:A-Ⅰ、C-Ⅲ_o、apoC-Ⅲ_s(s=1,2)、apoC's及可能的A-Ⅲ,几乎不含E和A-Ⅱ_o北京鸭HDL中apo的上述组成特点明显不同于易患动脉粥样硬化(As)的人及兔等动物,在一定程度上决定了北京鸭HDL的形成与代谢具有其特点,该特点与北京鸭不易形成As密切相关。     

Excitatory and inhibitory responses caused by norepinephrine in duck subfornical organ neurons are mediated by β- and α 2-adrenoceptor activation

The responsiveness of spontaneously active neurons in the subfornical organ (SFO) of adult ducks to angiotensin II (ANGII), norepinephrine (NE), isoproterenol (Iso, -agonist), phenylephrine (Phe, ₁-agonist) and clonidine (Clo, ₂-agonist) was investigated in brain slices with extracellular recording technique. 64% (n=90) of the neurons increased their activity after superfusion with ANGII, the rest were unresponsive. Application of NE activated 10 and inhibited 8 neurons (n=22); the excitation being correlated with an excitatory ANGII responsiveness of the same neurons and the inhibition with the absence of an ANGII responsiveness. Iso activated 74% (n=58) and Clo inhibited 88% (n=16) of the investigated neurons. Phe did not have an effect on the majority (60%) of the neurons and produced both excitatory and inhibitory actions on the remaining cells. These results offer a plausible explanation for the dose dependent dipsogenic effect of Iso and the failure of NE to elicit dose dependent drinking, which can be explained by its dual, excitatory and inhibitory effect on SFO neurons. It is further concluded, that peripherally applied Iso exerts its dipsogenic action in high concentration by a direct excitatory effect on SFO neurons via the open blood brain barrier. Under physiological conditions, afferent neuronal input of still unknown origin might specifically modulate the activity of SFO neurons, because plasma concentrations of NE are probably not high enough to activate SFO neurons from the blood side of the blood brain barrier.Abbreviations ACSF artificial cerebrospinal fluid - ANGII angiotensin II - Clo clonidine - Iso isoproterenol - NE norepinephrine - NTS nucleus of the solitary tract - Phe phenylephrine - SFO subfornical organ     

Congruency of phylogenies derived from different proteins

Summary This communication examines the question of phylogenetic congruency- i.e., whether or not the branching order of evolutionary trees is independent of the protein studied. It was found that trees constructed for birds on the basis of immunological comparison of their transferrins, albumins, and ovalbumins agree approximately with a published tree based on the amino acid sequences of their lysozymesc. This congruency is especially noteworthy with respect to the phylogenetic position of the chachalaca, a Mexican bird classified on morphological grounds in the family Cracidae of the order Galliformes. At the protein level, this species differs as much from non-cracid galliform birds as does the duck, which belongs to another order. Despite the organismal similarity between cracid and non-cracid galliform birds, the molecular relationship is remote. If this contrast between organismal and molecular results had been based on comparative studies with only lysozyme, one could have ascribed the contrast to the possibility that chachalaca lysozyme was paralogous, rather than orthologous, to the other bird lysozymesc. Examination of several proteins is thus desirable in cases of possible paralogy.This work was supported in part by grants GB-42028X from NSF and GM-21509 from NIH     

Expression of Insulin-Like Growth Factor System Genes in Liver Tissue During Embryonic and Early Post-Hatch Development in Duck (Anas platyrhynchos Domestica)

The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth, whereas the liver is the principal source of circulation IGF-I. In the present study, expression of IGF-I, IGF type-I receptor (IGF-IR), and IGF binding protein (IGFBP)-3 genes was quantified by RT-PCR in the liver tissue on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7 days post-hatching (PH) in meat-type Gaoyou ducks and egg-type Jinding ducks. The results showed that IGF-I mRNA could be detected as early as on E 13d, but the expression level was low throughout embryonic development before increasing dramatically by E 27d and 7 days PH in both duck breeds. However, Gaoyou ducks exhibited higher IGF-I mRNA level than Jinding ducks, and the differences were significant on E 13d, E 21d, and at 7 days PH. Expression of IGF-IR in liver increased gradually in the former stages of the embryonic development, reaching its highest point on E 21d, and then declined up until 7 days PH. The expression pattern of IGFBP-3 gene was similar to that of IGF-IR gene, increasing significantly from E 17d. The expression peak appeared on E 25d, then declined significantly just prior to hatching (day 27) and was followed by an increase at 7 days PH. In general, the expression level of IGF-IR and IGFBP-3 genes in Jinding ducks was higher than that in Gaoyou ducks. Inverse relationships were observed for the expression of IGF-I and IGF-IR, and IGF-I and IGFBP-3, whereas a positive relationship was observed for the expression of IGF-IR and IGFBP-3. Our data indicate a differential expression of selected genes that comprise the IGF system in the duck liver tissue during embryonic and early PH growth and development.     

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Porcine circovirus type 2 (PCV‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV‐2 capsid protein (CP) from plants is an essential first step towards the goal of a plant‐produced PCV‐2 vaccine candidate. In this study, the PCV‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV‐2 VLPs elicited specific antibody responses to PCV‐2 CP. This is the first report describing the expression of PCV‐2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.     

应用高效体积排阻色谱偶联多角度激光散射鉴定猪圆环病毒2型灭活疫苗及病毒样颗粒疫苗抗原

本文旨在建立基于高效体积排阻色谱(high-performance size-exclusion chromatography,HPSEC)偶联多角度激光散射仪(multi-angle laser light scattering,MALLS)的猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗抗原检测方法。以纯化的PCV2灭活病毒及病毒样颗粒(virus-like particles,VLP)为参照,对4家生产企业的2种PCV2灭活病毒疫苗(a、b)及VLP疫苗(c、d)破乳后进行HPSEC-MALLS检测及分子量分析;结合PCV2抗原检测卡、Western blotting和透射电子显微镜(transmission electron microscope,TEM),鉴定了特征色谱峰;考察了方法的重复性和检测线性。结果表明,两家企业生产的PCV2灭活病毒疫苗破乳液水相经HPSEC分离,在保留时间约13.3 min处出现抗原特征峰;MALLS计算该色谱峰分子量分别为2.61×10⁶(±4.34%) Da和2.40×10⁶(±2.51%) Da。两种VLP疫苗也在13.3 min处出现抗原特征峰,分子量分别为2.09×10⁶(±2.94%) Da和2.88×10⁶(±11.85%) Da,接近PCV2的理论分子量;同时在保留时间约11.4 min处也出现色谱峰,经检测分子量为4.37×10⁶(±0.42%) Da,TEM表征显示为VLP二聚体。取疫苗d和PCV2 VLP纯品进行重复检测,抗原色谱峰面积的RSD(n=3)均小于1.5%,重复性好;将PCV2 VLP纯品梯度稀释检测,VLP及其多聚体的色谱峰面积与浓度均呈良好的线性关系,R²分别为0.999及0.997,能够满足定量及多聚体含量分析。该方法有望成为一种准确、高效的PCV2疫苗的体外评价方法,用于质量评价与提升。