透析培养

本讨论了有效利用透析技术从发酵液中及时转移低分子杂质混合物,从而获得高密度发酵细胞的方法,章从反应系统、工艺策略、膜相关性能、应用我、生产性放大等方面说明了利用透析技术以达到高浓度细胞发酵的有效性和可靠性。透析技术不仅克服了微孔过滤和超滤中存在的膜孔堵塞弊端,而且如果应用“营养分离”补策略,还可以防止营养物质的损失而使培养基被高效利用,在实验条件下,透析培养的潜力通过两种反应模型进行演示：内置     

Enhanced calcium transients in glial cells in neonatal cerebellar cultures derived from S100B null mice

S100B is the major low-affinity Ca(2+)-binding protein in astrocytes. In order to study the role of S100B in the maintenance of Ca(2+) homeostasis, we generated S100B null mice by a targeted inactivation of the S100B gene. Absence of S100B expression was demonstrated by Northern and Western blotting for S100B mRNA and protein, respectively, and immunoperoxidase staining of sections of various brain regions. S100B null mice were viable, fertile, and exhibited no overt behavioral abnormalities up to 12 months of age. On the basis of light microscopy and immunohistochemical staining, there were no discernable alterations in the distribution and morphology of astrocytes or neurons in sections of adult brains of these mice. Astrocytes in cerebellar cultures derived from 6-day-old S100B null mice exhibited enhanced Ca(2+) transients in response to treatment with KCl or caffeine. On the other hand, granule neurons, in the same cultures, exhibited normal Ca(2+) transients in response to treatment with KCl, caffeine, or N-methyl-d-aspartate. These results demonstrate a specific decrease in Ca(2+)-handling capacity in astrocytes derived from S100B null mice and suggest that S100B plays a role in the maintenance of Ca(2+) homeostasis in astrocytes.     

Electrically evoked release of [(3)H]noradrenaline from mouse cultured sympathetic neurons: release-modulating heteroreceptors

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting alpha(2)-autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [(3)H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M(2) muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB(1) receptors. Prostaglandin E(2), sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E(2), and somatostatin were abolished (at the highest concentration of WIN 55, 212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the beta(2)-adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.     

Tetanus Toxin Enhances Protein Kinase C Activity Translocation and Increases Polyphosphoinositide Hydrolysis in Rat Cerebral Cortex Preparations

Abstract: Tetanus toxin (TeTx) has been recently demonstrated to be a Zn²⁺-dependent endopeptidase that cleaves synaptobrevin, a protein in part responsible for neurotransmitter release. Nevertheless, certain aspects of TeTx action, for example, the causal relationship between TeTx and protein kinase C (PKC; EC 2.7.1.37) activity cannot be explained by this cleavage alone. In the present study, primary neurons from fetal rat brain, synaptosomes, and whole slices have been used to examine this issue. Low doses of TeTx (≤ 10^?8M) caused PKC activity translocation in a manner similar to that produced by 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA (≤ 10^?7M) caused sustained PKC activity translocation, whereas TeTx produced translocation followed by relocation, depending on the dose and time of exposure. Immunoidentification with a monoclonal antibody recognizing both α and β isoforms revealed that TeTx induced moderate losses of PKC in the cytosolic fraction, without a comparable increase in the particulate fraction. Although moderate losses of activity were also noticed in the cytosolic fraction, the inconsistency with respect to activity translocation may be explained by translocation of additional PKC isoforms that are not identified by the antibody. Comparable levels of water-soluble inositol phosphate-labeled intermediates were obtained after treatment of cerebral cells and/or cortical brain slices with TeTx. Significant increases of 19 and 114% in the water-soluble myo-[2-³H]inositol-labeled inositol phosphate metabolites were found in cerebral cell culture and brain slices, respectively, after treatment with 10^?8M TeTx. TeTx (10^?8M) increased to the same degree the water-soluble inositol phosphate levels as did serotonin (10^?5M) or carbachol (10^?6M). It is suggested that part of the signaling cascade of TeTx consists of a component involving inositol phospholipid hydrolysis, which is associated with PKC activity translocation.     

Migration Behavior of Rodent Granule Neurons in the Presence of Antibody to the 4C5 Antigen

Abstract: We have reported the production of monoclonal antibody 4C5, which recognizes a cell surface antigen, the 4C5 antigen, involved in granule cell migration processes. In the present study, we investigated in a more precise manner the role of the 4C5 antigen in the different types of granule cell migrations that take place during cerebellar development. When cerebellar explant cultures derived from 10-day-old rats were performed for 2 days in the presence of monoclonal antibody 4C5, vertical granule cell migration, occurring in the presence of glia, was not significantly inhibited. In contrast, when monoclonal antibody 4C5 was included in the medium of microexplant cultures derived from 4-day-old mice and maintained for 4 days in vitro, granule cell migrations that occurred both parallel and perpendicular to the neurite bundles that were free of glia were inhibited. Moreover, a stronger inhibitory effect of the antibody was observed on migration perpendicular to the neurite bundles compared with the parallel type of migration. Our results indicate that the 4C5 antigen differentially affects the different developmental stages and types of granule cell migration during rodent cerebellar development.     

Responsiveness to hormone, growth factor and drug treatment of a human breast cancer cell line: Comparison between early and late cultures

Summary Growth rate, morphology, and responsiveness to mitogenic stimuli and pharmacological treatments were evaluated in early and late cell passages derived from the same clone of the widely used MCF-7 human breast adenocarcinoma cell line. Our results indicate dissimilarities between early (E) and late (L) passages for some of the parameters analyzed. The cells that underwent many subcultivations grew faster than the others; both appeared homogeneous in size and shape. The E cells, subcultured for almost 1 yr, displayed higher sensitivity to the mitogenic action of both estradiol, according to the level of estrogen receptor, and insulin-like growth factor-I than did the L cells, kept in culture for more than 10 yr. Cell responsiveness to two drugs, a novel steroid antiestrogen and a polysulfonated distamycin A derivative, was more pronounced in the early cultures only at the longer time of exposure to the higher concentration of the estrogen antagonist. In addition, a drug-induced inhibition of insulin-like growth factor-I binding to its receptor was shown in both E and L cells, the latter being less sensitive than the former when exposed to the antiestrogen. Finally, MCF-7 E and L cells showed similar behavior when drug-induced apoptosis was tested.     

Expression of extensin genes is dependent on the stage of the cell cycle and cell proliferation in suspension-cultured Catharanthus roseus cells

To isolate cDNAs expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cDNA library prepared from the S-phase cells of synchronized cultures of Catharanthus roseus. Sequence analysis shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins. Protein sequences deduced from the two cDNAs contain the characteristic pentapeptide repeat sequence, Ser-Pro-Pro-Pro-Pro, which is commonly observed in extensins. The protein sequences also share several other extensin characteristics such as the presence of a N-terminal signal peptide and a high content of Tyr and Lys residues. When C. roseus cell suspension cultures were synchronized by phosphate starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during the S and G2 phases of the cell cycle. However, significant amounts of the mRNAs also accumulated in phosphate-starved cells arrested in the G1 phase. In asynchronous cultures, both genes were expressed during the stationary phase, when cell proliferation ceased. The observed patterns of expression suggest that the extensin genes, cyc15 and cyc17, are under two types of regulation: one that depends on the stage of the cell cycle and another that is induced during the growth arrest. Thus, the products of these genes may function both during the progression through the cell cycle and in the strengthening of the cell wall after cell division.     

In vitro interactions between bone marrow stromal cells and hippocampal slice cultures

Bone marrow stromal cells (BMSCs) are capable of differentiating into various cell types including brain cells. Several groups have also demonstrated trophic effects of MSC grafts in experimental ischemia models. However, the underlying molecular mechanisms of these effects are not fully understood. We developed an “in vitro graft model” which consisted in a coculture of GFP-expressing BMSCs and hippocampal organotypic slice cultures. Total marrow cells (MCs) or BMSCs after one (BMSC^1P) or five passages (BMSC^5P) were transplanted on hippocampal slices. During the 10 days of our experiments, MCs and BMSC^1P migrated toward the tissue, but their total number remained constant. Conversely, the number of BMSC^5P decreased over the 10 days of the experiment, and no migration could be detected. Using immunohistochemistry, we observed that the hippocampal slices induced the expression of neural antigens in very few grafted cells, but MCs and BMSC^1P improved the conservation of the hippocampal slice culture. Similar experiments using BMSC^5P did not produce any significant change. We conclude that the number of passages greatly influence BMSCs survival rate, migration and neuroprotective capacities.     

Excitable properties of insect neurones in culture: A developmental study

The passive and excitable electrical properties of cockroach neurones growing in vitro have been investigated using intracellular recording techniques. The resting membrane potentials of the neurones are similar to those of their in vivo counterparts but the input resistances and membrane capacitive properties are more typical of embryonic insect neurones. During the first 12 days of growth in vitro the neurones exhibit delayed rectification in response to the injection of depolarising current steps. After this period “all or none” action potentials can be evoked by depolarising pulses in approximately half of the neurones tested. These spikes are abolised by 1 μM tetrodotoxin but are unaffected by 5 mM Co²⁺. Spontaneous excitatory activity develops in approx 25% of the neurones after 3 weeks in culture.