Influence of different ammonium,lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.     

酿酒酵母组氨酸转运蛋白Hip1p的泛素化水平对组氨酸利用的影响

【目的】为了研究泛素化对组氨酸转运及调控的影响。【方法】应用泛素化位点预测及定点突变等技术手段,Hip1p的3个潜在的泛素化位点K30、K42和K52被突变。这些突变的Hip1p被克隆到泛素化检测质粒中,检测泛素化位点突变对Hip1p的泛素化水平的影响。同时这些突变对细胞生长及组氨酸利用的影响也进一步做了检测。【结果】Hip1p的3个赖氨酸位点K30、K42和K52突变能有效降低其泛素化水平。同时,双重突变对其泛素化水平有明显的协同作用,并进一步影响了细胞生长和组氨酸利用。【结论】泛素化水平调控能有效调节组氨酸代谢,引起细胞对组氨酸利用的改变,为进一步研究氨基酸转运蛋白的调控机制提供了重要的依据。     

Effects of extracellular matrix on the malignant phenotype

Extracellular matrix molecules, including collagen, glycosaminoglycans (usually linked to a protein core as proteoglycan), elastin, and glycoproteins, influence the initiation and maintenance of differentiation of a variety of cell types. These molecules bind to the cell surface at specific sites and nonspecifically by electrostatic forces. Such interactions may alter the cell's response to growth and differentiation factors. After neoplastic transformation, most cells retain some dependence on these factors. This paper reviews the influence of matrix components on the phenotype of a variety of malignant cells and concludes that in vitro studies of malignant cell behavior require the utilization of an appropriate microenvironment.     

Role of auxin and gibberellin in the synthesis of Ascorbic Acid and growth of tissue explants

Coleoptile and root tips ofTriticum aestivum cv. Arnej 624 and those ofAvena sativa cv. Victory (Svalöf) as well as dry excised embryos ofTriticum aestivum cv. Rival (Svalöf) and those ofArachis hypogaea cv. 34 3A. H. were cultivated in media containing various concentrations of sucrose and growth regulators, like ascorbic acid, indole-3-acetic acid and gibberellin. Growth, differentiation and water uptake of the various explants were determined at regular time intervals. Further, the concentration of the endogenous ascorbic acid in mg./g. fresh weight, as well as the amount of this growth regulator utilized as per cent of the total were determined. Although all the three growth regulators promote growth in the explants, their effect is best felt when sucrose of a higher concentration (1.0 per cent) is added to the medium. In fact, the response to 1.0 per cent sucrose is sometimes as good as a combination of a growth regulator with sucrose, especially in the case of root explants. The results clearly indicate that the biosynthesis of ascorbic acid in the explants is catalyzed by the addition of indole-3-acetic acid as well as gibberellin. Simultaneously, the utilization of ascorbic acid is also appreciably increased by the presence of these growth regulators. Addition of 1.0 per cent sucrose to the medium containing the above mentioned growth regulators augments to a considerable extent not only the concentration of ascorbic acid, but also steps up its utilization. Enhancement of ascorbic acid as well as its increased utilization are correlated with rapid imbibition of water, growth and differentiation. The role of ascorbic acid in growth is discussed; and on the basis of the data presented here it is postulated that: (1) auxin and gibberellin function in the growth process by catalyzing the biosynthesis of ascorbic acid; and (2) that ascorbic acid not only participates in activation of various enzyme systems, but also stimulates the production of adenosine triphosphate by acting as an electron donor in photosynthetic phosphorylation as well as oxidative phosphorylation; (3) that the above action of ascorbic acid creates a favourable redox balance for synthesis of nucleic acids, proteins, enzymeproteins, and cell-wall constituents, thus enabling the processes of cell division and enlargement to proceed at a fast rate; and (4) that the relative rates of cell division and cell enlargement as well as “ageing” will determine the pattern of plant development.     

Protein kinase CK2 signal in neoplasia

Protein kinase CK2 (previously known as casein kinase II) is a protein serine/threonine kinase that has been implicated in cell growth and proliferation. The focus of this review is on the apparent role of CK2 in cancer. Studies from several laboratories have shown a dysregulated expression of the kinase in tumors. Nuclear matrix and chromatin appear to be key sites for signaling of the CK2 activity in relation to cell growth. Several types of growth stimuli produce a common downstream response in CK2 by enhancing its nuclear shuttling. The neoplastic change is also associated with changes in intracellular localization of the kinase so that a higher nuclear localization is observed in tumor cells compared with normal cells. Experimental studies suggest that dysregulated expression of the alpha subunit of CK2 imparts an oncogenic potential in the cells such that in cooperation with certain oncogenes it produces a profound enhancement of the tumor phenotype. Recent studies have provided evidence that overexpression of CK2 in tumor cells is not simply a reflection of tumor cell proliferation alone but additionally may reflect the pathobiological characteristics of the tumor. Of considerable interest is the possibility that CK2 dysregulation in tumors may influence the apoptotic activity in those cells. Approaches to interfering with the CK2 signal may provide a useful means for inducing tumor cell death.     

应用系统理论的方法建立套作玉米系统的数学模型

一、前言 目前,系统理论越来越广泛地深入地应用到各个科学研究和生产领域,甚至社会科学的范围。通常我们最简单地用一个方框图表示一个系统(图1)。     

Latent transforming growth factor-beta binding proteins (LTBPs)--structural extracellular matrix proteins for targeting TGF-beta action

Growth factors of the transforming growth factor-beta family are potent regulators of the extracellular matrix formation, in addition to their immunomodulatory and regulatory roles for cell growth. TGF-beta s are secreted from cells as latent complexes containing TGF-beta and its propeptide, LAP (latency-associated peptide). In most cells LAP is covalently linked to an additional protein, latent TGF-beta binding protein (LTBP), forming the large latent complex. LTBPs are required for efficient secretion and correct folding of TGF-beta s. The secreted large latent complexes associate covalently with the extracellular matrix via the N-termini of the LTBPs. LTBPs belong to the fibrillin-LTBP family of extracellular matrix proteins, which have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. Currently four different LTBPs and two fibrillins have been identified. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures. LTBPs are now known to exist both as soluble molecules and in association with the extracellular matrix. An important consequence of this is LTBP-mediated deposition and targeting of latent, activatable TGF-beta into extracellular matrices and connective tissues. LTBPs have a dual function, they are required both for the secretion of the small latent TGF-beta complex as well as directing bound latent TGF-beta to extracellular matrix microfibrils. However, it is not known at present whether LTBPs are capable of forming microfibrils independently, or whether they are a part of the fibrillin-containing fibrils. Most LTBPs possess RGD-sequences, which may have a role in their interactions with the cell surface. At least LTBP-1 is chemotactic to smooth muscle cells, and is involved in vascular remodelling. Analyses of the expressed LTBPs have revealed considerable variations throughout the molecules, generated both by alternative splicing and utilization of multiple promoter regions. The significance of this structural diversity is mostly unclear at present.     

Stoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates.

Metabolic by-product secretion is commonly observed in oxygen-limited cultures. Oxygen limitations occur because of limits in the capacity of the respiratory system or because of the oxygenation limits of the cultivation method used. The latter restriction is of considerable practical importance since it results in a critical cell concentration above which oxygenation is insufficient, leading to by-product secretion. In this study we used a flux balance approach to determine optimal metabolic performance of Escherichia coli under variable oxygen limitations. This method uses linear optimization to find optimal metabolic flux patterns with respect to cell growth. Cell growth was defined as precursor requirements on the basis of a composition analysis. A growth-associated maintenance requirement of 23 mmol of ATP per g of biomass and a non-growth-associated maintenance value of 5.87 mmol at ATP per g (dry weight)-h were incorporated on the basis of a comparison with experimental data. From computations of optimal growth increased oxygen limitations were found to result in the secretion of acetate, formate, and ethanol in that order. Consistent with the experimental data in the literature, by-product secretion rates increased linearly with the growth rate. The computed optimal growth under increasing oxygen limitation revealed four critical growth rates at which changes in the by-product secretion pattern were observed. Concomitant with by-product secretion under oxygen limitations were changes in metabolic pathway utilization. The shifts in metabolism were characterized by changes in the metabolic values (computed as shadow prices) of the various redox carriers. The redox potential was thus identified as a likely trigger that leads to metabolic shifts.2+ ă     

Metabolism characteristics of <Emphasis Type="Italic">Chelativorans oligotrophicus</Emphasis> by two-phase growth on the mixture of EDTA and glucose

The obligate destructor of ethylene diamine tetraacetate—a culture of Chelativorans oligotrophicus LPM-4—did not grow on a medium with glucose, but it was good to use it under cultivation on a mixture with EDTA after considerable decrease of the EDTA concentration in the medium (two-phase growth). Strong inhibition of hexokinase and glucose 6-phosphate dehydrogenase in cell exracts 4 mM EDTA was revealed. Using EDTA, cells accumulated polyphosphates whose rate decreased during glucose utilization phase. High activities of polyphosphate biosynthesis ferments (adenylat kinase and polyphosphate kinase) were distinguished during the first phase of the cultivation; considerable decrease of them and increase of polyphosphate glucokinase were found during the second phase of the cultivation. This points to the possible participating of polyphosphates in glucose metabolism as a supplementary energy source.     

The induction of serine/threonine protein phosphorylations by a PDGFR/TrkA chimera in stably transfected PC12 cells

Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.     

Contributions of atmospheric CO and hydrogen uptake to microbial dynamics on recent Hawaiian volcanic deposits

A series of sites were established on Hawaiian volcanic deposits ranging from about 18 to 300 years old. Three sites occurred in areas that supported tropical rain forests; the remaining sites were in areas that supported little or no plant growth. Sites >26 years old consumed atmospheric CO and hydrogen at rates ranging from about 0.2 to 5 mg of CO m(-2) day(-1) and 0.1 to 4 mg of H(2) m(-2) day(-1), respectively. Respiration, measured as CO(2) production, for a subset of the sites ranged from about 40 to >1,400 mg of CO(2) m(-2) day(-1). CO and H(2) accounted for about 13 to 25% of reducing equivalent flow for all but a forested site, where neither substrate appeared significant. Based on responses to chloroform fumigation, hydrogen utilization appeared largely due to microbial uptake. In contrast to results for CO and hydrogen, methane uptake occurred consistently only at the forest site. Increasing deposit age was generally accompanied by increasing concentrations of organic matter and microbial biomass, measured as phospholipid phosphate. Exoenzymatic activities (acid and alkaline phosphatases and alpha- and beta-glucosidases) were positively correlated with deposit age in spite of considerable variability within sites. The diversity of substrates utilized in Biolog Ecoplate assays also increased with deposit age, possibly reflecting changes in microbial community complexity.     

Effect of surfactant pluronic F-68 on CHO cell growth, metabolism, production, and glycosylation of human recombinant IFN-γ in mild operating conditions

The control of glycosylation to satisfy regulatory requirements and quality consistency of recombinant proteins produced by different processes has become an important issue. With two N-glycosylation sites, γ-interferon (IFN-γ) can be seen as a prototype of a recombinant therapeutic glycoprotein for this purpose. The effect of the nonionic surfactant Pluronic F-68 (PF-68) on cell growth and death was investigated, as well as production and glycosylation of recombinant IFN-γ produced by a CHO cell line that was maintained in a rich protein-free medium in the absence or presence of low agitation. Under these conditions, a dose-dependent effect of PF-68 (0-0.1%) was shown not only to significantly enhance growth but also to reduce cell lysis. Interestingly, supplementing the culture medium with PF-68 led to increased IFN-γ production as a result of both higher cell densities and a higher specific production rate of IFN-γ. If cells were grown with agitation, lack of PF-68 in the culture medium decreased the fraction of the fully glycosylated IFN-γ glycoform (2N) from 80% to 65-70% during the initial period. This effect appeared to be due to a lag phase in cell growth observed during this period. Finally, a global kinetic study of CHO cell metabolism indicated higher efficiency in the utilization of the two major carbon substrates when cultures were supplemented with PF-68. Therefore, these results highlight the importance of understanding how media surfactant can affect cell growth as well as cell death and the product quality of a recombinant glycoprotein expressed in CHO cell cultures.     

Buoyant density, growth rate, and the cell cycle in Streptococcus faecium.

The buoyant density in rapidly growing Streptococcus faecium 9790 cells varies over the cell cycle, in contrast to the density in Escherichia coli. Buoyant density in S. faecium was measured by using Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.) density gradients. We found that the mean and coefficient of variation of the population density increased with growth rate; and within a population, the mean cell volume, which was measured electronically, increased with density. These results were compared with electron microscopic measurements of the size distributions of cell wall growth sites within each fraction of the density gradient. As the density increased within a population, the frequency of large cells increased and the frequency of newly initiated cell wall growth sites increased. These effects were more marked as the growth rate increased. Next, these data were regrouped by cell size by using the size of the central growth site as an index of cell cycle stage. Each frequency value was weighted by the proportion of the population represented by that density fraction. Then, the average buoyant density was calculated for each value of cell size. In all cell populations, the density decreased and then increased as the central site enlarged. Peripheral growth sites were initiated as density reached a maximum. At faster growth rates, density increased more steeply, and new peripheral growth sites opened up at a higher frequency. We suggest that the rate at which density increases during the cell cycle correlates with the initiation of new cell wall growth sites.     

Differential utilization of pyrene as the sole source of carbon by Bacillus subtilis and Pseudomonas aeruginosa strains: role of biosurfactants in enhancing bioavailability

AIMS: Our goal is to compare the efficiency of utilization of pyrene as the sole source of carbon for growth and energy by two nonactinomycetous groups of bacteria viz., Bacillus subtilis DM-04 and Pseudomonas aeruginosa mucoid (M) and nonmucoid (NM) strains, isolated from a petroleum-contaminated soil sample of north-east India. METHODS AND RESULTS: Bacillus subtilis DM-04 and P. aeruginosa M and NM bacterial strains were capable of secreting biosurfactant in the culture medium while growing on pyrene and their pyrene utilizing efficiency was demonstrated by correlating the bacterial growth in the presence of pyrene as the sole source of carbon along with a concomitant decrease in pyrene content from the culture medium with respect to time. The biosurfactant secreted by the respective bacterial strains enhanced the apparent solubility of pyrene by factors of 5-7 and influenced the bacterial cell surface hydrophobicity resulting in higher uptake and utilization of pyrene by bacteria. The growth of B. subtilis DM-04 and P. aeruginosa M and NM strains at the expense of pyrene after 96 h showed an assimilation of about 48.0 +/- 1.1% (mean +/- SD) and 32.0 +/- 0.6% (mean +/- SD) of pyrene carbon, respectively, showing differences in metabolism of pyrene by these bacterial strains. CONCLUSIONS: Bacillus subtilis DM-04 strain exhibited higher utilization and cellular assimilation of pyrene compared with P. aeruginosa M and NM strains. Further, the biosurfactants produced by the bacteria under study are capable of enhancing the solubility of pyrene in aqueous media and can influence the cell surface hydrophobicity of the biosurfactant-producing strains that results in a higher uptake of pyrene. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be suggested that the bacteria used in this study are suitable candidates for practical field application for effective in situ bioremediation of pyrene-contaminated sites.     

Inter-species differences in maximum specific growth rates and cell yields of bifidobacteria cultured on oligosaccharides and other simple carbohydrate sources

The abilities of seven bifidobacterial isolates ( Bifidobacterium adolescentis , B. bifidum (two strains), B. catenulatum , B. infantis , B. longum , B. pseudolongum ) to utilize 15 different carbohydrate sources (eight oligosaccharide products, and a variety of monosaccharides and disaccharides) were studied, with regard to maximum specific growth rates and production of bacterial cell mass. Results showed that substrate utilization was highly variable and that considerable interspecies and interstrain differences existed. Galactooligosaccharides and oligofructose, with a low degree of polymerization, supported best growth of the test micro-organisms. In contrast, xylooligosaccharides and pyrodextrins were almost invariably poor bifidobacterial substrates. In many species, maximum specific growth rates and bacterial cell yields were higher on oligosaccharides compared to their monosaccharide constituents, particularly with respect to fructooligosaccharides. Bifidobacterium pseudolongum , B. longum and B. catenulatum were the most nutritionally versatile isolates studied in relation to the range of oligosaccharide products utilized, and the extent to which bacteria could grow on these substrates.     

Analysis of initiation of sites of cell wall growth in Streptococcus faecium during a nutritional shift

Three-dimensional reconstruction methods were applied to electron micrographs of Streptococcus faecium to study the initiation of cell wall growth sites during a nutritional shift experiment. Upon lowering the mass doubling time from 76 to 33 min by the addition of excess glutamate, the formation of new cell wall growth sites accelerated above the old steady-state rate at about the same time (10 to 15 min) as did mass, RNA, protein, cell numbers, and autolytic capacity but considerably before DNA (30 min) and peptidoglycan (20 min) synthesis did. During the shift, the average range of cell volumes over which new wall growth sites were introduced did not change significantly. However, upon the shift there was an increase in the frequency of cells having new sites, which was due to the faster-growing cells initiating more new sites in peripheral locations before division. After a transition period, the number of new sites per milliliter of culture increased at a rate that paralleled that of the culture mass. These findings support a model in which new sites are introduced when cells grow to a relatively constant, growth rate-independent size, while the rate at which sites form and grow increases with the growth rate. In this model, chromosome synthesis does not regulate the formation of new sites of cell wall growth, but existing sites cannot be completed until rounds of chromosome synthesis are completed.     

The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli

The bacteria for which there is evidence that proteins of the ParAB family act in chromosome segregation also undergo developmental transitions that involve the ParAB homologues, raising the question of whether the partition activity is equivalent to that of plasmid partition systems. We have investigated the role in partition of the parAB locus of a free-living bacterium, Pseudomonas putida, not known to pass through developmental phases. A parAB deletion mutant, compared with wild type, showed slightly higher frequencies of anucleate cells in exponentially growing cultures but much higher frequencies in deceleration phase. This increase was growth medium dependent. Oversupply of ParA and ParB proteins also raised anucleate cell levels, specifically in the deceleration phase, in wild-type and mutant strains and regardless of medium, as well as generating abnormal cell morphologies. Absence or oversupply of ParAB function had either slight or considerable effects on growth rate, depending on temperature and medium. The need for the Par proteins in chromosome partition thus appears to be subject to the cell's physiological state. Three sequences similar to cis-acting stabilization sites of Bacillus subtilis are present in the P. putida oriC-parAB region. One was inserted into an unstable mini-F and shown to stabilize it in E. coli in a ParAB-dependent manner.