依尼奥小单孢菌抗性基因sisR的克隆研究

从产西索米星的依尼奥小单孢菌中克隆高抗性新基因sisR,借助计算机设计PCR引物,从西索米星的产生菌依尼奥小单孢菌的染色体DNA中,经PCR扩增获得DNA序列长度不等的DNA片段。将这些DNA片段克隆至pUC19载体质粒并导入大肠杆菌,从中筛选到5个抗西索米星的转化子(其中一个命名为sisR)显示对西索米星的高抗性(超过1000μg/mL)。经DNA测序并通过互联网Biast比对,确认对该抗性负责的DNA片段是一个未见报道的新基因。     

Atomic Structural Models of Fibrin Oligomers

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Effect of methylmercuric chloride (MMC) on fibrin polymerization

Methylmercuric chloride (MMC) in concentrations 0.1–10μM reduces the amount of fibrinopeptides released from thrombinactivated human fibrinogen. However, the fibrin clot formation is not discriminated and the turbidity of the fibrin gel is even augmented. MMC does not cause such changes in the process of repolymerization of fibrin monomers. The addition of fibrinopeptides to the fibrin monomers results in a similar increase of turbidity of the repolymerizing sample in the presence of MMC as in the case of fibrinogen clotting. These experiments indicate that MMC modifies the structure of fibrin in the presence of fibrinopeptides.     

Anti-fibrin antibody binding in valvular vegetations and kidney lesions during experimental endocarditis.

In Streptococcus sanguinis (sanguis) induced experimental endocarditis, we sought evidence that the development of aortic valvular vegetation depends on the availability of fibrin. Endocarditis was induced in New Zealand white rabbits by catheter placement into the left ventricle and inoculation of the bacteria. Fibrin was localized in the developing vegetation with 99mTechnetium (Tc)-labeled anti-fibrin antibody one or three days later. When rabbit anti-fibrin antibody was given intravenously on day 1, the mass of aortic valvular vegetation was significantly reduced at day 3; infusion of non-specific rabbit IgG showed no effect. The 99mTc-labeled anti-fibrin antibody also labeled kidneys that showed macroscopic subcapsular hemorrhage. To learn if the deposition of fibrin in the kidneys was a consequence of endocarditis required a comparison of farm-bred and specific pathogen-free rabbits before and after the induction of endocarditis. Before induction, the kidneys of farm-bred rabbits were labeled, but specific pathogen-free rabbits were free of labeling and signs of macroscopic hemorrhage. After 3 days of endocarditis, kidneys of 10 of 14 specific pathogen-free rabbits labeled with 99mTc-labeled anti-fibrin antibody and showed hemorrhage. Kidney lesions were suggested to be a frequent sequellae of S. sanguinis infective endocarditis. For the first time, fibrin was shown to be required for the continued development of aortic valvular vegetations.     

Adherence of Candida species to fibrin clots in vitro

The adherence of six Candida species to fibrin clots was studied using a simple, in vitro technique. Yeast suspensions were incubated with fibrin clots and the number of adherent organisms quantified as follows: after washing, the clots were subjected to vortex mixing and the number of CPU's which subsequently grew on Sabourauds medium were counted. Adhesion was directly proportional to the concentration of Candida species in the suspension (r=0.99 p<0.001). C. albicans and C. tropicalis exhibited marked adherence whereas C. krusei, C. gulliermondi and C. glabrata adhered less readily. C. parapsilosis was intermediate in its ability to adhere.     

高分子人工血管材料大鼠肌肉内的急性期反应

本实验研究临床常用的几种人造血管生物材料在大鼠体内引起的急性期组织反应,并与自主研发的丝素蛋白改性聚氨酯(Silk fibroin-polyurethane(1:1),SF-PU(1:1))材料相比较,以期找出组织相容性最佳的材料。将涤纶(Dacron)材料、膨化聚四氟乙烯(Expanded polyterafluoroethylene,e-PTFE)材料、聚氨酯(Polyurethane,PU)材料、以及丝素蛋白改性聚氨酯复合材料(SF-PU(1:1))埋植入大鼠肌肉内,通过大鼠急性毒性实验、肌肉植入局部组织反应实验、局部组织切片染色、白细胞及血小板计数,探讨几种材料对大鼠的局部及全身影响,研究比较各组材料的组织相容性。结果表明:涤纶材料的组织相容性较差;其余各组材料的局部组织炎性反应较轻,且白细胞及血小板计数与假手术组无显著性差异。故认为涤纶作为临床上常用的人造血管材料组织相容性最差,所研发的SF-PU(1:1)材料及另两种临床上常用的e-PTFE材料和PU材料的组织相容性较好,尤以SF-PU(1:1)材料的组织相容性最好,结合SF-PU(1:1)优异的物理性能,在小口径人造血管的研制方面有很大的研究前景。     

Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network

The results of biochemical, immunochemical, and X-ray studies of the structures of fibrinogen and fibrin molecules were analyzed. The mechanisms of the successive formation of the fibrin three-dimensional network were described: the polymerization of monomeric molecules with the formation of bifilar protofibrils, the lateral association of protofibrils, and the embranchment of the forming fibrils. Data on the electron and confocal microscopy of the polymeric fibrin were considered. The role of the known polymerization centers of fibrin which participated in the formation of protofibrils and their lateral association was discussed. Data on the existence of the previously unknown polymerization centers were given. In particular, the experimental results demonstrated that one of such centers which participated in the formation of protofibrils was located in the Bβ12–46 fragment, and did not require the cleavage of fibrinopeptide B for its functioning. The results of the computer modeling of the spatial structure of the fibrin(ogen) molecule and the intermolecular interactions in the course of the fibrin polymerization were presented. The location of the αC domains in the fibrin(ogen) molecule and their role in the polymerization process were discussed. Information on the structure of the calciumbinding sites of fibrin(ogen) and the functional role of Ca²⁺ in fibrin polymerization was published. The structure of factor XIII(a) and the mechanisms of fibrin stabilization by this factor were briefly described.     

Truncated vitronectins: binding to immobilized fibrin and to fibrin clots, and their subsequent interaction with cells.

The plasminogen activator inhibitor-1 (PAI-1) is stabilized in its inhibitory conformation by binding to Vitronectin (Vn). The anchorage of PAI-1 to the fibrin fibers was recently shown to be mediated by Vn, and as such to modulate fibrinolysis. Here we report the mapping of the fibrin binding sites in Vn using truncated recombinant Vns, and show that two segments of Vn are involved: one at its carboxyl terminus (within residues 348-459) and one at its amino terminus (within residues 1-44). This mapping sets the stage for (i) the design of specific inhibitors for the Vn-fibrin interaction; (ii) for studying the role of this interaction in the anchoring of endothelial cells and platelets onto the fibrin clot; and (iii) for getting a deeper insight into the mechanism of the Vn-fibrin interaction in fibrinolysis. (c)2002 Elsevier Science.     

纤维蛋白胶存在的问题与相应的解决方法

分析了纤维蛋白胶存在的问题,如蛋白胶不同供体来源、成份来源、应用的简易性与有效性、止血功效局限、机械强度与纤溶抑制剂的选择等,并提出相应的解决方案。     

血浆纤维蛋白黏合剂的制备及其在重组肉中应用

目的:充分利用肉品加工中所产生的碎肉和屠宰中产生的大量血液制备纤维蛋白黏合剂.方法:采用纤维蛋白原冷沉淀法从肉用动物血浆制备了黏合剂,之后将其用于碎肉的加工中,制备重组肉制品及优化制备过程及参数,而且进一步对重组肉产品的粘合效果做了对比实验.结果:建立了高效的重组肉制备工艺,利用肉用动物血浆制备了纤维蛋白黏合剂,之后用其制备重组肉,并优化了制备过程及参数,制备工艺简单、周期短、成本低、自制纤维蛋白黏合剂所生产的重组肉性状好于其它黏合剂.结论:此方法操作简单,成本低廉,所制作的重组肉粘合效果好、口感好、绿色环保.     

SDS－聚丙烯酰胺凝胶电泳纤维蛋白自显影法检测不同分子量的纤溶酶原激活剂

ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）纤维蛋白显影方法是经ＳＤＳ－ＰＡＧＥ将不同分子量的纤溶酶原激活剂（ＰＡ）分开,然后再转溶纤维蛋白板使之产生溶带而检测ＰＡ物质活性及分子大小的方法。此法与Ｗｅｓｔｅｒｎ印迹方法比具有检测更简便、灵敏、快速的特点,且是活性检测,但其分子量分析精度不如Ｗｅｓｔｅｒｎ印迹法。本研究用ＳＤＳ－ＰＡＧＥ纤维蛋白自显影方法对本室表达的重组组织型－ｐＡ（ｒｔ－ＰＡ）及突变体Ｎｌｌ７Ｑ／Ｎｌ８４Ｑ／△（Ｋ２９６——Ｇ３０２）及标准尿激酶（ＵＫ）和ｒｔ－ＰＡ进行了分析鉴定。     

The Use of Monoclonal Antibodies for Studying the Fibrin Polymerization

Examples of using monoclonal antibodies (MAb) for studying the fibrin polymerization mechanism are considered. MAb with epitopes situated in the fibrin polymerization sites or in the recognition sites of enzymes thrombin, plasminogen, and factor XIII, which are the functional partners of fibrin, are primarily discussed. The MAb to epitopes in various regions of A, B, and polypeptide chains of the functionally important E, D, and C domains of fibrin are successively described.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.     

Exogenous fibrin matrix precursors stimulate the temporal progress of nerve regeneration within a silicone chamber

The silicone chamber model permits the investigation of the cellular and molecular events underlying successful regeneration of the rat sciatic nerve across a 10 mm gap. When 25 l chambers are implanted prefilled with phosphate-buffered saline (PBS), it takes 5–7 days before sufficient fibrin matrix (derived from plasma precursors) accumulates naturally to form a complete bridge across the chamber gap; at 1 week postimplantation, cellular migration into the matrix from the nerve stumps is just beginning. The temporal progress of regeneration might be stimulated if a fibrin matrix, conductive to cell migration, was provided to the nerve stumps at or shortly after the time of chamber implantation. To test this hypothesis, chambers were prefilled, at the time of implantation, with different preparations of homologous plasma. A solution of 90% platelet-free plasma dialyzed against PBS (DP) formed a fibrin matrix by 24 hours postimplantation that, like the naturally formed matrix, had a predominantly longitudinal orientation. The temporal progress of regeneration was stimulated in the DP-prefilled chambers; at 17 days postimplantation, the extents of Schwann cell migration and axonal elongation were significantly greater than in the control system. In contrast, prefilling chambers with either non-citrated plasma or DP + calcium resulted in the generation of a matrix within 8 minutes that was composed of randomly oriented fibrin polymers. These matrices significantly retarded the progress of regeneration.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Polymerization Sites in the D-Domain of Human Fibrin(ogen)

The present work deals with localization of previously unknown polymerization sites of the fibrin DD-fragment. D-dimer we obtained has a pronounced inhibitory effect on fibrin polymerization (IC₅₀ = 0.06 M). The inhibitory effect of the D-fragment disappeared after reduction and carboxymethylation. However, polypeptide chains _DD (B134-461) and _DD (63-411)₂ of the DD-fragment, isolated by preparative electrophoresis, displayed their inhibitory activity. For instance, the rates of fibrin protofibril lateral association were decreased twice in the presence of _DD and _DD chains at their molar ratios to fibrin of 0.40 and 0.15, respectively. The IC₅₀ values for _DD and _DD were 0.24 and 0.10 M, respectively. Highly specific inhibition of protofibril lateral association suggests that the protofibril lateral association sites are located in B134-461 and 63-411 regions of the fibrin D-domain. Our data confirm those reported by Doolittle et al. regarding the -chain and a hypothesis about -chain of fibrin D-domain (Yang, Z., Mochalkin, I., and Doolittle, R. F. (2000) Biochemistry, 97, 14156-14161).     

The extraction effect of ultrasound during plasma clot disruption

The products of the plasma clot destruction by the low-frequency ultrasound (US) were analyzed using the combination of SDS gel-electrophoresis, gel filtration chromatography and scanning electron microscopy. It was found that US (27 kHz) did not cause activation of the plasmin system or covalent bonds cleavage in the fibrin molecules. At US intensities less than 21.6 W/cm² there was extraction of blood serum proteins, which are located in the pores of the fibrin network. The increase in intensity of ultrasonic action resulted in protofibril dissociation, which was accompanied by further release into the solution of the blood serum proteins, located inside fibrin fibers. After US cavitation protein extracted from the plasma clot underwent aggregation. Interaction between free protofibrils resulted in formation of insoluble fibrin particles.     

Stable hepatocyte transplantation using fibrin matrix

Fibrin matrix, a naturally derived biodegradable polymer matrix, was evaluated as a scaffold for hepatocyte transplantation in an athymic mouse model. One week after transplantation, opaque conglomerates of the transplanted hepatocytes and fibrin matrix were found on the intestinal mesentery, whereas no transplanted hepatocytes were observed in control groups (transplantation of hepatocytes suspended in culture medium). The hepatocytes in the conglomerates retained hepatocyte-specific functions, as examined with histochemical and immunohistochemical stainings. Stable hepatocyte engraftment may thus be achieved by hepatocyte transplantation using fibrin matrix.