Homogeneous interferon-inducing 22K factor is related to endogenous pyrogen and interleukin-1

In vitro stimulation of mononuclear cells from human peripheral blood with mitogens causes the release of factors (monokines and lymphokines) which possess distinct biological activities. One such factor, termed 22K, can induce production of human beta-interferon (HuIFN-beta) in cultured human fibroblasts, thereby rendering these cells resistant to virus infection. Here we report the complete purification and partial sequencing (39 N-terminal amino acids) of this factor, whose relative molecular mass was estimated by SDS-polyacrylamide gel electrophoresis to be 17,000 (17K). In addition to an antiviral effect, the pure protein exhibits several other biological activities. Most significantly, intravenous (i.v.) injection of the factor in rabbits caused fever and granulopenia at doses of 0.1-1 microgram per kg, effects which we attribute to a monokine called endogenous pyrogen (EP). In vitro, the protein was scored as positive in a LAF (lymphocyte-activating factor) assay at 0.1-1 ng ml-1. LAF and EP are considered to be members of one family of monokines, called interleukin-1 (IL-1). For this reason, and also because the amino-acid sequence of the 22K factor is at least partially homologous to a complementary DNA-derived IL-1 sequence, we postulate that the 22K factor also belongs to the IL-1 family.     

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.     

Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.     

Basic fibroblast growth factor fused to a signal peptide transforms cells

Basic fibroblast growth factor (bFGF) is a potent growth and angiogenic factor that is found in abundance in tissues such as brain, hypothalamus, kidney and cartilage. Despite this copious production of bFGF, most of these tissues are not undergoing either active growth or angiogenesis, suggesting that bFGF activity must be regulated so as to prevent autostimulation of cell growth. In cultured cells, bFGF is associated mainly with cells and basement membranes and is not released into the medium. Prevention of release could be a mechanism for regulation of bFGF activity and may be a consequence of the apparent absence of a secretory-signal sequence in the bFGF protein. Here we investigate whether this regulation can be overridden through the forced secretion of bFGF. Such secretion might provide the bFGF access to its receptor and in turn lead to autocrine transformation of the cell. We report that bFGF, as specified by a recombinant plasmid, is itself unable to induce such transformation, but acquires this ability after fusion with a secretory-signal sequence. The resulting transformants undergo unusual morphological alteration and display tumorigenicity.     

Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin

Fibroblast growth factors (FGFs) are a large family of structurally related proteins with a wide range of physiological and pathological activities. Signal transduction requires association of FGF with its receptor tyrosine kinase (FGFR) and heparan sulphate proteoglycan in a specific complex on the cell surface. Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity. Although crystal structures of binary complexes of FGF-heparin and FGF-FGFR have been described, the molecular architecture of the FGF signalling complex has not been elucidated. Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide. The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain. The structure of the FGF1-FGFR2-heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling.     

Suppression of experimental glomerulonephritis by antiserum against transforming growth factor beta 1

Glomerulonephritis is an inflammation of the kidney characterized by the accumulation of extracellular matrix within the damaged glomeruli, impaired filtration and proteinuria. In its progressive form, the disease destroys kidney function leading to uraemia and death, unless dialysis therapy or kidney transplantation is available. The pathogenesis of glomerulonephritis is incompletely understood, but the eliciting factor is thought often to be an immunological injury to mesangial and/or other resident cells in the glomeruli. We have used an animal model of acute mesangial proliferative glomerulonephritis to show that this disease is associated with increased production and activity of transforming growth factor beta 1 (TGF-beta 1), an inducer of extracellular matrix production. Here we report that administration of anti-TGF-beta 1 at the time of induction of the glomerular disease suppresses the increased production of extracellular matrix and dramatically attenuates histological manifestations of the disease. These results provide direct evidence for a causal role of TGF-beta 1 in the pathogenesis of the experimental disease and suggest a new approach to the therapy of glomerulonephritis.     

重组人碱性成纤维细胞生长因子的纯化及生物活性鉴定

目的 :探讨重组人碱性成纤维细胞生长因子 (rhbFGF)的纯化工艺和活性鉴定 .方法 :将rhbFGF工程菌大量扩增后通过包涵体提取、复性、阳离子交换、凝胶过滤等技术进行纯化 .结果 :纯化后的rhbFGF ,相对分子质量为 17× 10 3,蛋白纯度为 95 %以上 ,比活为 1 7× 10 6 U/mg ,对NIH3T3细胞具有明显的促分裂活性 .结论 :通过复性和两步层析的纯化方法可获得高纯度、高回收率和高生物活性的rhbFGF ,可用于实验室研究及临床试验     

利用双启动子质粒在毕赤酵母中高效表达人FGF1

基于p PIC9K构建了同时含有AOX1和FLD1的双启动子质粒,并成功转入毕赤酵母GS115中,建立了一个新型的全长人活性FGF1表达系统。测序结果和SDS-PAGE显示质粒构建成功,两种启动子可同时被甲醇诱导。液质联用分析表明,目的蛋白的氨基酸组成与人源性FGF1基本一致,且在生物活性测定中表现出良好的生物相容性。经过大规模发酵与凝胶过滤层析,重组FGF1蛋白的产量为197 mg/L,约为当前报道的最高表达水平的两倍,且纯度达到97%。     

重组人酸性成纤维细胞生长因子的遗传毒性

目的:了解重组人酸性成纤维细胞生长因子(rhaFGF)皮肤外用的遗传毒性。方法:用CHL细胞染色体畸变试验、小鼠骨髓PCE微核试验和Am es试验方法对rhaFGF进行遗传毒性研究。结果:rhaFGF各剂量组和阴性对照组在加或不加S9情况下,其CHL染色体畸变率均<5%;各剂量组的骨髓细胞微核细胞率与阴性对照组比较均差异无显著性(P>0.05);rhaFGF各剂量组(0.5~5 000μg/皿)对TA97、TA98、TA100、TA102菌珠在加或不加S9混合液条件下均无致突变作用。结论:在所选试验和剂量范围内未见到rhaFGF具有遗传毒性。     

碱性成纤维细胞生长因子对无血清培养大鼠生长板软骨细胞的影响

目的：探讨碱性成纤维细胞生长因子（bFGF）用于无血清培养扩增软骨细胞的可行性及最适质量浓度。方法：分离、培养大鼠肋生长板软骨细胞（rat costochondral growth plate chondrocyte,RGC）。细胞化学和免疫细胞化学染色的方法检测第1代RGC中蛋白多糖和Ⅱ型胶原的表达。观察0．1、1．0、10．0和100．0μg/L bFGF对无血清培养RGC形态的影响,并分别用^3H—TdR和^3H—Proline掺入法检测上述质量浓度bFGF对无血清培养RGC增殖和胶原合成的影响。结果：第1代RGC表达蛋白多糖和Ⅱ型胶原,保持了软骨细胞的分化表型。随着bFGF质量浓度的增加RGC的形态由多角形变为梭形和圆形。与无血清对照组相比,上述4个质量浓度的bFGF均显著促进RGC的增殖和胶原合成（P〈0．05）,其中1．0和10．0μg／L bFGF促RGC增殖的作用与10％胎牛血清（FBS）对照组差异无显著性意义（P〉0．05）,但促胶原合成作用较弱,相当于10％FBS对照组的印％左右（P〈0．05）。结论：bFGF可用于无血清扩增软骨细胞,1．0～10．0μg/L是适宜的质量浓度范围,但其促胶原合成作用较弱,还需添加其他因子以弥补促胶原合成作用的不足。     

柞蚕核型多角体病毒fgf基因的克隆和分析

为获得柞蚕NPV的全基因组序列,在柞蚕尸体中分离纯化柞蚕NPV,提取其基因组DNA,分别建立ApNPV DNA的HindⅢ和SalⅠ基因文库．对插入片段进行测序,经过同源性分析发现一个与人源fgf基因相似的基因,其读码框含有185aa．核苷酸和氨基酸同源性比较的结果表明：在genebank中没有同源性极高的序列,是个新基因;柞蚕NPV的fgf基因与云杉卷叶蛾NPV的同源性较高,分别为56．1％和45．9％,而与家蚕NPV及AcNPV的同源性相对较低,说明柞蚕NPV与家蚕NPV在进化上亲缘关系较远．该基因的克隆为进一步研究其在病毒感染过程中的作用打下基础．     

Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo

Cutting the axons of the cholinergic neurons that project to the hippocampal formation results in death of most of these cells. Previous studies have shown that administration of nerve growth factor before or at the same time as the lesion will prevent this cell death. Here we demonstrate that basic fibroblast growth factor (FGF) administered into the brain reduces the death of cholinergic neurons in the medial septum and diagonal band of Broca after transection of their axons, in both young adult and aged rats. Moreover, FGF can partially protect against death of cholinergic neurons even when administered two days after axonal transection. These results indicate a possible function for FGF in the normal support of basal forebrain cholinergic neurons, but its range of activity could be wider, for FGF also supports noncholinergic neurons in vitro, it is localized in many of the central nervous system neurons, and it is found in relatively high concentrations in the brain.     

FGF-21对2型糖尿病大鼠心肌氧化应激影响的研究

目的探讨成纤维细胞生长因子-21（FGF-21）对2型糖尿病大鼠心肌氧化应激的影响。方法用高热量脂肪饮食联合小剂量链脲佐菌素建立2型糖尿病大鼠模型,随机分成2型糖尿病组与FGF-21治疗组,并与正常对照组比较。测定三组问大鼠血浆及心肌组织葡萄糖糖、胰岛素、FGF-21、脂质产物及氧化应激指标的变化。结果2型糖尿病组血糖、胰岛素、游离脂肪酸（FFAs）、甘油三酯、FGF-21及氧化应激指标平均水平较其他两组显著增高（P〈0．05）。FGF-21治疗组血糖、胰岛素、FFAs、甘油三酯及氧化应激指标平均水平较2型糖尿病组显著降低（P〈0．05）,与对照组无差异。结论FGF-21可以改善2型糖尿病大鼠心肌心肌氧化应激状态,并参与调控2型糖尿病代谢紊乱。     

碱性成纤维细胞生长因子对骨髓基质细胞的生物学效应

目的探讨碱性成纤维细胞生长因子(bFGF)对骨髓基质细胞(BMSC)粘附、增殖和分化等生物学效应的影响.方法利用体视学计数、MTT法及ALP试剂盒分别测定不同浓度bFGF诱导一定时间后BMSG的粘附特性、增殖和分化情况的变化.结果10n/mLbFGF明显促进BMSG的粘附,但是200ng/mLbFGF反而不利于细胞粘附;在细胞增殖和分化测定中,100ng/mLbFGF明显促进细胞增殖,但细胞碱性磷酸酶含量也最低.结论碱性成纤维细胞生长因子对骨髓基质细胞的生物效应是复杂和多方面的,可以作用于骨髓基质细胞的粘附、增殖和分化等多个环节,这种影响与碱性成纤维细胞生长因子的剂量有关.     

Structure of a beta1-adrenergic G-protein-coupled receptor

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.     

Structure of the apoptotic protease-activating factor 1 bound to ADP

Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.