培养基组分对沙打旺(Astragalus adsurgens Pall)组培根增殖的影响及其培养滤液提取物的化感活性

采用L9 (315 )正交设计,研究了B5培养基营养组分对沙打旺组培根增殖的影响;并采用玻璃皿滤纸培养法,对其培养滤液提取物进行生物测定以验证沙打旺组培根的化感活性.结果显示:培养基的所有营养组分中,Fe2 对沙打旺组培根增殖的影响最大,蔗糖、H2PO 4、 Mg2 、 Mn2 、 Cu2 、 Zn2 、 BO3-3、 Co2 、 I-、C8H12ClNO3 C12H18Cl2N4OS C6H5O2N C6H12O6的影响次之,氮、Ca2 、MoO2-4 和NAA的影响最小.根据不同养分条件下沙打旺组培根干重的极差分析,筛选出适宜沙打旺组培根快速增殖的优化培养基.培养滤液提取物的生物测定结果表明沙打旺组培根培养过程中可能产生化感物质;化感作用强度的差异预示营养胁迫可能影响其化感物质的产生.研究为沙打旺组培根再生与繁殖提供一定依据,并揭示养分条件可能是该植物表达化感作用的影响因素.     

细胞外钙调素对烟草悬浮培养细胞蛋白质磷酸化作用的影响

用液体闪烁计数法研究了细胞外钙调素对烟草悬浮培养细胞质蛋白质磷酸化的作用。结果表明烟草细胞细胞质蛋白质磷酸化活性在细胞培养过程中逐渐增加,达到最高峰后又开始下降。在细胞质蛋白质磷酸化强度高峰时,加入抗CaM血清后,细胞质蛋白质磷酸化活性受到了部分抑制。加抗CaM血清后再补加CaM能够部分解除抗CaM血清对细胞质部分与细胞核部分蛋白质磷酸化的抑制作用。外加纯化钙调素可以引起烟草悬浮培养细胞细胞质蛋白质磷酸化的活性增强,并且这种增强作用具有时间（高峰为70min）与剂量（最适为CaM10-7mmol/L）依赖性。CaM引起的细胞质蛋白质磷酸化变化与红光所引起的细胞质蛋白质磷酸化变化在时间进程上是不相同的。     

Separation and characterization of proteins in rice (Oryza sativa) suspension cultured cells

Proteins extracted from suspension cultured cells of rice were separated by two-dimensional polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 103 electroblotted proteins were analyzed. The N-terminal amino-acid sequences of 20 out of 103 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Internal amino-acid sequences of 32 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. Furthermore, the concanavalin A-peroxidase method was used to determine which of the 103 proteins were glycosylated, and in vitro and in vivo phosphorylation was carried out to identify some of the phosphorylated proteins. Using this experimental approach, we could identify the major proteins involved in growth and development of rice cell suspension cultures and discuss on the physiological function of some of these identified proteins including the calcium binding protein, superoxide dismutase and rice ascorbate peroxidase. This revised version was published online in June 2006 with corrections to the Cover Date.     

骨骼发育中的转录因子Cbfa1

骨骼由骨和软骨共同构成.最近的研究表明,转录因子Cbfa1不仅控制骨的形成和生长,还影响软骨组织成熟,并且可能与破骨细胞分化和软骨血管侵入有关.     

肠道菌群变化对肠上皮细胞Myd88蛋白及炎症因子的影响

目的 研究肠道菌群变化对体外培养的正常大鼠肠黏膜上皮细胞Myd88蛋白水平及其下游TNF-α等炎症因子的影响。方法 选取10只健康大鼠,实验室适应性喂养5 d后继续常规喂养7 d并处死,取结肠组织进行细胞培养得到原代肠黏膜上皮细胞,将所得细胞分为5组,分别用正常细胞培养液（A组）、正常大鼠肠腔菌溶液（B组）、正常大鼠黏膜菌溶液（C组）、溃疡性结肠炎大鼠肠腔菌溶液（D组）、溃疡性结肠炎大鼠黏膜菌溶液（E组）培养,通过蛋白质印迹法检测各组细胞中Myd88蛋白含量,通过酶联免疫吸附法检测TNF-α和IL-6含量。结果 D组、E组与A组比较,细胞中Myd88蛋白及TNF-α、IL-6水平显著增高（Ps＜0.01）,D、E组与B、C组比较Myd88蛋白及TNF-α、IL-6水平亦显著增高（Ps＜0.01）,而B组、C组相较于A组差异无统计学意义（Ps＞0.05）。进一步比较D组与E组、B组与C组间Myd88蛋白及TNF-α、IL-6水平差异亦无统计学意义（Ps＞0.0.5）。结论 （1）肠道菌群结构的改变可引起肠黏膜细胞Myd88通路的激活及炎症因子的释放。（2）肠道中细菌种类、数量、分布位置等因素的综合变化与肠黏膜炎症反应的发生关系密切。     

Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010. © 2010 Wiley‐Liss, Inc.     

The time-pattern of rises and falls in proliferation fades with senescence of mortal lines and is perpetuated in immortal rat hepatoma fao cell line

Summary Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3–4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22–23 additional population doublings each. Normal embryonic fibroblasts culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attribution and repair processes regulates the waves of proliferation. Equal contributors to these studies.     

Indole-3-acetic acid production by bacteroids from soybean root nodules

Purine nucleotide and RNA synthesis have been investigated at the different growth stages of carrot ( Daucus carota L.) cells grown in suspension cultures. At the early growth stages an increase in the content of RNA was observed, although at later stages RNA was degraded. The highest rates of incorporation of [¹⁴C]-labelled adenosine into ATP and GTP were observed at the late growth sttages. This indicated that purine slavage was more importnt at the late growth stages, while de novo synthesis was dominant during the initial growth stages. This pattern was also reflected by increased levels, in the cell dividison phase, of theenzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amido-transferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cells in the stationary phase, that were starved for sucrose and phosphate, showed a dramatic increase in cellular metabolism, as judged from a rapid uptake and incorporation of [³²P]-labelled phosphate into nucleotides and RNA, when incubated in fresh medium.     

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300–1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35 000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60°C) and resistant to inactivation by trypsin (2 h, 37°C, 10 μg/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.