Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

人类新基因WDR24的研究初报

WD40家族是一类结构保守、功能复杂的蛋白.目前很多研究显示该家族成员通过参与MAPK信号途径调控细胞内信号转导而影响细胞的基本生命活动.为了鉴定参与细胞生命活动的新基因,运用同源基因克隆法,通过PCR技术扩增获得一个新的人类基因WDR24, 其cDNA全长3 302 bp,2 373 bp长的开放阅读框编码由790个氨基酸残基组成的蛋白质.生物信息学分析表明,WDR24蛋白在进化上高度保守,与其他脊椎动物中的同源蛋白组成了一个功能未知的亚家族.蛋白序列分析显示其中有6个WD40重复序列和1个ERK的停泊位点D-domain.RT-PCR分析表明,该基因在所有被检测的人类胚胎组织中表达.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins,Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we''ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.¹     

辐射增敏剂甲硝唑氨酸对坪期V_(79)细胞DNase活力的影响

研究了辐射增敏剂甲硝唑氨酸(CM)对受照前后坪期V_(79)细胞DNase活力的影响。结果表明CM在一定浓度范围内(1—10mmol/L)对DNase的激活作用有浓度依赖关系。细胞经6—12Gyγ线照后DNase活力亦增高,若照射合并CM处理后,则对DNase激活有相加作用。还就DNase活力升高与DNA链断裂间的可能关系进行了讨论。     

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Trans-stilbene oxide, trans-β-methylstyrene, 7,8-oxide, trans-β-ethylstyrene, 7,8-oxide, trans-β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his⁻ strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10⁶ of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.     

Biosynthesis and metabolic engineering of glucosinolates

Summary. Glucosinolates are amino acid-derived natural plant products found throughout the Capparales order. Glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi. The conversion of amino acids to aldoximes is a key step in glucosinolate biosynthesis. This step is catalyzed by cytochromes P450 from the CYP79 family. The post-aldoxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional group and low substrate-specificity for the side chain. Therefore, we have been able to metabolically engineer new glucosinolate profiles into Arabidopsis by altering the levels of endogenous CYP79s and by introducing exogenous CYP79s. The approach has great potential for design of metabolically engineered plants with improved pest resistance and increased nutritional value. Received December 7, 2001 Accepted December 21, 2001