Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis

The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.     

Tumors producing human tumor necrosis factor induced hypercalcemia and osteoclastic bone resorption in nude mice

We used a Chinese hamster ovary cell line that had been transfected with the human tumor necrosis factor (TNF) gene and constitutively produced TNF when transplanted in nude mice to study the effects of continuous production of TNF on calcium homeostasis. Continuous exposure to TNF caused increased osteoclastic bone resorption and humoral hypercalcemia in these animals. The mice bearing TNF-producing tumors were significantly hypercalcuric compared to mice bearing control tumors, but urinary cAMP excretion was unchanged. Mice bearing Chinese hamster ovary cell tumors containing the empty vector did not demonstrate hypercalcemia or increased bone resorption. This model system using transfected cells to continuously produce cytokines in vivo is more analogous to the pathophysiological conditions present in patients than intermittent injections and can produce much longer exposures than infusion pumps. Such model systems should allow a better understanding of the role of factors involved in humoral hypercalcemia.     

Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor-alpha (TGF-alpha) is synthesized by a variety of tumor cell lines and stimulates osteoclastic bone resorption in vitro. The mechanism by which TGF-alpha increases osteoclast activity is unknown. We used a human marrow culture system that forms osteoclast-like multinucleated cells (MNCs) to determine the effects of recombinant human TGF-alpha on MNC formation. Addition of 0.01 ng/ml TGF-alpha for the 1st week followed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for the subsequent 2 wk significantly increased MNCs. Treatment of these cultures with TGF-alpha without later addition of 1,25(OH)2D3 did not increase MNC formation. Autoradiographic studies revealed that TGF-alpha stimulated proliferation of precursors for MNCs, and 1,25(OH)2D3 increased their rate of fusion into MNCs. Addition of murine epidermal growth factor (EGF) (0.1 ng/ml) followed by 1,25(OH)2D3 also significantly stimulated MNC formation. These data suggest that TGF-alpha and EGF may stimulate bone resorption by increasing the proliferation of osteoclast precursors, which leads to increased numbers of osteoclasts.     

大鼠脂肪间充质干细胞的成骨分化

目的:观察大鼠脂肪间充质干细胞经成骨诱导向成骨细胞分化的生物学特性,探讨其作为骨组织工程种子细胞的可行性。方法:实验于2004-07/2006-03在中南大学湘雅医院中心实验室完成主要工作。①取健康SD大鼠双侧腹股沟区脂肪垫,消化法分离出脂肪间充质干细胞,接种入含有体积分数为0.1的新生牛血清的低糖DMEM培养基进行原代培养。②取第3代的脂肪间充质干细胞,用含有体积分数为0.1的新生牛血清、0.1μmol/L地塞米松、50μmol/L抗坏血酸、10mmol/Lβ-甘油磷酸钠的高糖DMEM培养基诱导其向成骨细胞分化。③于3,5,7,10,12,14,21d分别采用倒置显微镜观察细胞形态及增殖情况、Gomori改良钙钴法碱性磷酸酶染色、茜素红S钙结节染色和Ⅰ型胶原免疫细胞化学染色检测脂肪间充质干细胞成骨分化的情况。结果:①脂肪间充质干细胞原代细胞呈成纤维细胞样长梭形外观,传代稳定,细胞形态均一。②经成骨诱导,脂肪间充质干细胞体积增大,呈多角形;成骨诱导14d,Gomori改良钙钴法碱性磷酸酶染色,细胞胞浆内可见浅棕色至棕黑色的颗粒,平均染色阳性率为80%;碱性磷酸酶活性随时间的延长而逐渐增高[3,5,7,10,12,14d依次为(2.43±0.09),(3.60±0.08),(5.01±0.09),(7.75±0.07),(9.59±0.09),(10.94±0.10)μkat/L];成骨诱导21d,钙结节形成明显,茜素红S染色,呈红色结节;成骨诱导7d,Ⅰ型胶原免疫细胞化学染色,细胞胞浆呈棕黄色,胞核经苏木精复染为蓝色。结论:大鼠脂肪间充质干细胞经成骨诱导具有成骨细胞的生物学特性,可作为骨组织工程的种子细胞。     

Survey on pretransfusion testing

BACKGROUND: Hospitals and blood centers throughout the United States use a variety of reagents and methods to perform pretransfusion testing. A survey was developed to determine the reagents and methods in use and their relative prevalence in different work settings. STUDY DESIGN AND METHODS: A national survey on pretransfusion testing was conducted. Surveys were distributed to state and regional blood bank associations, which then distributed them to hospitals and blood centers within their region. In most instances, the blood centers distributed the survey to the local hospitals. Completed surveys were returned to the authors for review, and all information was entered into a database for analysis. RESULTS: Analysis of the data shows that the majority of blood banks use monoclonal reagents for ABO testing and monoclonal-polyclonal blended reagents for Rh testing. The data show that anti-IgG and polyclonal antihuman globulin reagents are used almost equally for antibody screening (detection) tests and that most blood banks use a three-cell antibody-screening test. Slightly more than 50 percent of hospitals use an immediate-spin crossmatch in the absence of unexpected antibodies. CONCLUSION: A number of approved reagents and methods are used by blood bank laboratories for pretransfusion testing. Facility size (number of beds) and type tend to influence the choice of methods and reagents employed. This survey provides an opportunity for blood bank laboratories to compare their current practices with those of their peers.     

Prolonged procoagulant activity on overstretch-injured coronary arteries in pigs

Summary.  This study was designed to assess the time course and nature of the vascular procoagulant response after 1.5-fold balloon overstretch injury of the coronary arteries in pigs. Arteries were excised for chromogenic assay of bound factor (F)Xa and thrombin at 24 h, 3 days, 1 week, or 2 weeks after injury. FXa at the site of injury remained elevated for 1 week (4.9 ± 5.9 µg cm⁻², n  = 10), compared with non-injured control arteries (0.4 ± 0.2 µg cm⁻², n  = 18, P  = 0.00025), while thrombin was increased only at 24 h. Tissue factor protein was abundant in non-injured coronaries (10 ± 6 ng µg⁻¹ total protein, n  = 9) and levels were unchanged by injury (13 ± 11 ng µg⁻¹, n  = 6) or 24-h administration of tissue factor pathway inhibitor (16 ± 6 ng µg⁻¹, n  = 6). Persistent tissue factor-mediated procoagulant activity may explain the need for prolonged anticoagulation to attenuate neointimal formation after balloon-induced coronary injury.