发酵工程

912901 一种极端嗜卤原始细菌对烃的降解[英]/Bertrand,J.C.…∥Lett.Appl.Microbio1.-1990,11(5).-260～263[译自DBA,1991,10(3),91-01267] 从盐沼地分离到的原始菌株EH4能降解饱和烃和芳香烃.用二种方法探讨了生长对氯化钠的需求.第一组实验取不同浓度(2.

The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demon strated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior ob- served during development and pathogenesis.

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepato- cytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxan- induced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/ progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/ progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.

Effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells

Objective： We investigated the effects of intermittent negative pressure on osteogenesis in human bone marrowderived stroma cells （BMSCs） in vitro. Methods： BMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently （pressure： 50 kPa, 30 min/times, and twice daily）. The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast mi- croscopy, the determination of alkaline phosphatase （ALP） activities, and the immunohistochemistry of collagen type 1. The mRNA expressions of osteoprotegerin （OPG） and osteoprotegerin ligand （OPGL） in BMSCs were analyzed by real-time polymerase chain reaction （PCR）. Results： BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure, the activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the mRNA expression of OPG increased significantly （P〈0.05） and the mRNA expression of OPGL decreased significantly （P〈0.05） after 2 weeks, compared with the control. Conclusion： Intermittent negative pressure could promote osteogenesis in human BMSCs in vitro.

Osteogenic potential of rabbit marrow stromal stem cells cultured in vitro: a hi stochemical and scanning electron microscopic study

To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.