Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Proteolytic Processing of Chromogranin B and Secretogranin II by Prohormone Convertases

Abstract: Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH₃ cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.     

Synthesis of the segment (11–23) located in the first tandem repeat of plasma kallikrein: comparative binding studies of this and another segment (328–343) to high-molecular-mass kininogen

The synthesis of porcine plasma kallikrein (pPK) segment (11–23), of sequence Phe-Phe-Arg-Gly-Gly-Asp-Val-Ser-Ala-Met-Tyr-Thr-Pro, present in the first tandem repeat sequence of the regulatory chain of PK, has been accomplished following the peptide fragments (5 + 4 + 4) condensation strategy in solution, as well as by fluorenylmethoxycarbonyl solid-phase chemistry. This and another synthetic PK segment of residues (328–343) present in the fourth tandem repeat sequence [Cys(ACM)-Ser-Leu-Arg-Leu-Ser-Thr-Asp-Gly-Ser-Pro-Thr-Arg-Ile-Thr-Tyr] and synthesized by a solid-phase method, were fully characterized by ¹H nuclear magnetic resonance, fast atom bombardment mass spectrometry, amino acid composition and reversed-phase high-performance liquid chromatography. Proteolysis of these peptides by either rat PK (rPK) or trypsin resulted in cleavages between Arg↓Gly for pPK (11–23) and between Arg↓Leu and Arg↓Ile for rPK (328–343). Kinetic studies revealed that for peptide pPK (11–23), the catalytic efficiency (k_cat/K_m) of rPK is 9-fold higher than that of trypsin, but for the other peptide, rPK (328–343), k_cat/K_m of trypsin is 49-fold higher than that of rPK. The facile cleavage of pPK (11–23) by rPK confirms the Arg¹³↓Gly¹⁴ position as the site of autolytic degradation of PK and also explains its special preference for Phe-Phe-Arg sequence.     

中药夏枯草对人甲状腺癌细胞系SW579增殖周期及凋亡的影响

目的:探讨夏枯草对人甲状腺癌细胞系SW579细胞生长的抑制作用及其对细胞增殖周期和凋亡的影响。方法:采用甲基噻唑(MTT)比色法和生长曲线测定不同浓度夏枯草在不同作用时间内对在体外培养SW579细胞增殖的影响,同时应用流式细胞术检测细胞增殖周期及凋亡率的变化。结果:夏枯草可在G0/G1期阻滞人甲状腺癌细胞系SW579的增殖,使S期细胞比率降低;在一定范围内,夏枯草的浓度越高、作用时间越长,对肿瘤细胞生长的抑制作用越强,凋亡率也越高。结论:夏枯草能抑制人甲状腺癌细胞系SW579细胞生长,并诱导细胞凋亡而阻止细胞周期。     

硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。     

Syntheses of argininal semicarbazone containing peptides and their applications in the affinity chromatography of serine proteinases

Eight argininal semicarbazone containing peptides prepared by liquid phase synthesis were all found to be reversible inhibitors of model serine proteinases including trypsin and plasma kallikrein (PK). Among the peptides tested, those having a Lys residue at position P2 displayed the maximum binding potency towards PK. One of the peptides, Leu-enkephalin-argininal semicarbazone, a comparatively weak inhibitor, was chosen in order to develop an affinity-based purification protocol for PK. The affinity column was prepared by covalent attachment of the NH2-terminal moiety of the peptidyl semicarbazone to a solid-phase matrix bearing a spacer group. For efficient binding of PK, it was found necessary to optimize parameters like the concentration of inhibitor linked to the solid matrix, the ionic strength of the buffer used, the temperature and the pH. The majority of the bound enzyme could be recovered following elution with guanidine hydrochloride or benzamidine hydrochloride in a high salt buffer at pH 6.0. The usefulness of the affinity procedure towards the purification of other serine proteinases is also discussed.     

Loss of endothelial furin leads to cardiac malformation and early postnatal death

In mammals, seven proprotein convertases (PCs) cleave secretory proteins after basic residues, and four of them are called furin-like PCs: furin, PC5, PACE4, and PC7. In vitro, they share many substrates. However, furin is essential during development since deficient embryos die at embryonic day 11 and exhibit multiple developmental defects, particularly defects related to the function of endothelial cells. To define the role of furin in endothelial cells, an endothelial cell-specific knockout (ecKO) of the Furin gene was generated. Newborns die shortly after birth, indicating that furin is essential in these cells. Magnetic resonance imaging revealed that ecKO embryos exhibit ventricular septal defects (VSD) and/or valve malformations. In addition, primary cultures of wild-type and ecKO lung endothelial cells revealed that ecKO cells are unable to grow. Growth was efficiently rescued by extracellular soluble furin. Analysis of the processing of precursors of endothelin-1 (ET-1), adrenomedullin (Adm), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein 4 (BMP4) confirmed that ET-1, Adm, and TGF-β1 are in vivo substrates of endothelial furin. Mature ET-1 and BMP4 forms were reduced by ～90% in ecKO purified endothelial cells from lungs.     

Purification and sequence determination of bovine atrial natriuretic factor

We report the purification and the sequence determination of bovine atrial natriuretic factor (ANF) in acid extracts of bovine atrial appendages. The monitoring of the activity along the purification steps was performed with a radio-receptor assay using bovine adrenal cortex membranes sites and 125I ANF. Bovine ANF was separated by carboxymethyl agarose gel chromatography from catecholamines and major protein contaminants. It behaved as a 3 K dalton peptide on Sephadex G-50. The active fractions were then subjected to high performance liquid chromatography (HPLC) using a sulfopropyl cation exchange column. Subsequent purification steps by reverse phase mode on Ultrapore RPSC, Vydac TP 218 and uBondapak using acetonitrile gradients led to the obtention of a pure fraction which amino acid sequence was identical to that for human ANF. This confirms the high degree of homology of ANF structure among mammalian species and advocates the use of the radio-receptor assay based on bovine adrenal receptor for measuring human ANF.     

Ontogeny of a novel pituitary protein (7B2) in the human fetal intestine

The developmental profile of the concentration of a novel pituitary protein (7B2) was studied immunochemically in the human gastrointestinal tract from 12 weeks of gestation to 4 months after birth and was compared to the distribution in the adult. 7B2-like immunoreactivity (IR-7B2) was detected in all segments studied, but no gross changes were seen through fetal life. At term higher concentrations of IR-7B2 were found in the duodenum and the antrum, which is similar to the distribution of adult man. Gel permeation chromatography revealed that the main peak of 7B2 immunoreactivity in the fetal intestinal extract eluted with a Kav of 0.3. Similar elution profiles were also observed in extracts of human adult intestine.