Growth Dynamics of Mitochondria in Synchronized Chinese Hamster Cells

The increase in cell volume (from electronic cell sizing) and the apportionment of this volume amongst the nuclear, cytoplasmic, and mitochondrial subcellular compartments (from electron microscopy) were studied throughout the cell division cycle in partially synchronized cultures of Chinese hamster V79-S171 cells. Average whole cell volume was found to increase smoothly, consistent with the doubling in one generation of individual cell volume. Nuclear size increased in like fashion. Mean total mitochondrial volume and number of mitochondria per cell both showed a different kind of variation, most notably a significant decrease in G₁ and G₂ as compared with mid S. These results are therefore counter to a model of simple doubling of mitochondria either synchronously with the cell division cycle or asynchronously. Absolute mean values per cell for log phase Chinese hamster cells were also determined, as follows: whole cell volume, 710 μ³; nuclear volume, 190 μ³; total mitochondrial volume, 37.5 μ³; number of mitochondria per cell, 90.     

Growth of Enveloped RNA Viruses in a Line of Chinese Hamster Ovary Cells with Deficient N-Acetyl-glucosaminyltransferase Activity

Sindbis and vesicular stomatitis viruses were grown in a line (termed 15B) of Chinese hamster ovary (CHO) cells that is deficient in a specific UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase. Both viruses replicated normally in the cell line, but the glycoproteins of the released virus migrated faster on sodium dodecyl sulfate-polyacrylamide gels than did glycoproteins of virus grown in parent CHO cells. Digestion of the viral glycoproteins with Pronase followed by gel filtration demonstrated that the glycopeptides of Sindbis-15B virus were much smaller than the glycopeptides of Sindbis-CHO virus. In addition, Sindbis-15B viral glycopeptides but not Sindbis-CHO viral glycopeptides contained terminal α-mannose residues as shown by their susceptibility to α-mannosidase digestion. These findings demonstrate that the oligosaccharide units of the glycoproteins of vesicular stomatitis and Sindbis viruses are altered when the viruses are grown in 15B cells. We conclude that the N-acetylglucosaminyltransferase that is missing in 15B cells normally participates in the biosynthesis of the oligosaccharide units of the viral glycoproteins, and in the absence of this enzyme incomplete oligosaccharide chains are produced. Viruses released from 15B cells appear to retain full infectivity; Sindbis-15B virus, however, showed a significant decrease in hemagglutination titer compared with that of Sindbis-CHO virus.     

Life Cycle Analysis of Mammalian Cells: II. Cells from the Chinese Hamster Ovary Grown in Suspension Culture

A method for life cycle analysis in mammalian cells which utilizes the collection function has been applied to the Chinese hamster ovary grown in suspension. The following durations were found for the various parts of the life cycle: S, 4.13 hours; G1, 4.71 hours; G2, 2.81 hours; mitosis, 0.81 hours. The cell has a total generation time of 12.4 hours as opposed to 20.1 hours for the S3 HeLa cell. However, the relative lengths of each phase of the life cycle are identical within experimental uncertainty in the two cells.     

An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells

The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 μg 10⁻⁶ cells day⁻¹, one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-κB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation, was examined. Suppression of NF-κB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.     

Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work, such cultures were studied by both flow cytometry and conventional microbiological methods and were found to contain various numbers of viable cells. Pretreatment of such cultures with penicillin G, and subsequent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (±five) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a population of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) decreased from 70 to 27 h when the number of viable cells was increased from 30 to 10⁵ per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost constant (at some 20 h). Provided there were more than nine (±five) initially viable cells per flask, the number of initially viable cells did not affect the final percentage of resuscitable cells in the culture. The lag period could be ascribed in part to the time taken to restore the membrane permeability barrier of starved cells during resuscitation, as revealed by flow cytometric assessment of the uptake of the normally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4-[3-methyl-2, 3-dihydro-(benzo-1, 3-oxazole)-2-methylidene]-1-(3′-trimethylammonium propyl)-pyridinium diiodide}. Although cell populations which contained fewer than nine ±five viable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 X 10⁶) were able to accumulate rhodamine 123 after 80 to 100 h of incubation, showing the ability of a significant number of the cells in the population at least to display “metabolic resuscitation.” Resuscitation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logarithmic-phase M. luteus cultures as the resuscitation medium. We conclude that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remaining (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which they are capable of colony formation.     

Mitochondrial Thymidine Kinase 2 but Not Deoxyguanosine Kinase Is Up-Regulated During the Stationary Growth Phase of Cultured Cells

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.     

Studies of the Growth in Culture of Plant Cells

Mitochondria have been isolated from sycamore cells (Acer pseudoplatanusL.) grown in suspension culture, and resemble those of otherplant tissues. Malate, succinate, and NADH are oxidized withrespiratory control. The respiration is partially inhibitedby antimycin A and KCN, but not by amytal and rotenone. Octylguanidine,oligomycin, and uncouplers all affect the coupled respiration. The proportion of the respiration resistant to KCN was foundto change during the life of the culture, being greatest duringthe lag phase and least during the linear phase. The relationshipof these changes in the electron transport pathways to the changingdemand of the culture for phosphorylated and other intermediatesis discussed.     

1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture

We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.     

Agonist-induced Internalization of the Caenorhabditis elegans Muscarinic Acetylcholine Receptor GAR-3 in Chinese Hamster Ovary Cells

Many membrane-bound neurotransmitter receptors are known to be internalized by exposure to agonist. This agonist-induced receptor internalization is considered to play important roles in receptor-mediated signaling. Here we investigated the internalization of GAR-3, a Caenorhabditis elegans muscarinic acetylcholine receptor, using cultured mammalian cells. When Chinese hamster ovary cells stably expressing GAR-3 were treated with carbachol, GAR-3 was internalized in a dose- and time-dependent manner. Approximately 60% of the cell surface receptor was internalized by exposure to 1 mM carbachol for 1 h. Carbachol-induced GAR-3 internalization was suppressed by treatment with hypertonic sucrose, which blocks the formation of clathrin-coated pits. Overexpression of a dominant-negative dynamin mutant (DynK44A), but not of a dominant-negative β-arrestin mutant (Arr319–418), substantially inhibited carbachol-induced internalization of GAR-3. Thus, these data suggest that GAR-3 undergoes agonist-induced internalization via a clathrin- and dynamin-dependent but β-arrestin-independent pathway. Depletion of Ca²⁺ by simultaneous treatment of the cells with BAPTA/AM (Ca²⁺ mobilization blocker) and EGTA (Ca²⁺ influx blocker) almost completely blocked agonist-induced GAR-3 internalization. Moreover, treatment of the cells with the Ca²⁺ ionophore A23187 led to GAR-3 internalization in the absence of agonist. These results indicate that Ca²⁺ plays a critical role in GAR-3 internalization. We tested whether the third intracellular (i3) loop of GAR-3 is involved in agonist-stimulated receptor internalization. A GAR-3 deletion mutant lacking a large central portion of the i3 loop exhibited an internalization pattern comparable to that of the wild type, suggesting that the central i3 loop is not required for the internalization of GAR-3.     

中国田鼠卵巢细胞gpt基因自发和亚砷酸钠诱发突变的分子分析

本文研究了亚砷酸钠对CHO-AS52细胞gpt基因的致突变作用。实验结果表明,亚砷酸钠能诱发该基因发生突变,且其突变频率随砷浓度的增加而增高。PCR分析指出,绝大多数亚砷酸钠诱发的CHO-AS52突变体的gpt基因完全缺失。在CHO-AS52细胞自发的、50μmol/L和100μmol/L亚砷酸钠诱发的突变体中,gpt基因完全缺失者所占比率分别为36.00％、54.72％及66.67％。对亚砷酸钠诱发的非缺失型gpt基因突变的PCR产物直接进行DNA序列分析表明,在9个突变细胞克隆中,有2个发生移码突变,其余7个突变细胞克隆的gpt基因结构未发现改变,碱基的改变可能发生在基因启动子区。     

The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates

To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.     

Effects of Sugars on the Glucosylation of Exogenous Hydroquinone by Catharanthus roseus Cells in Suspension Culture

The effects of sugars on the glucosylation of exogenous hydroquinone(HQ) was investigated by supplying individual sugars simultaneouslywith HQ to a suspension culture of Catharanthus roseus cells.The production of arbutin was enhanced as much as 2- to 3-foldby sucrose or glucose at concentrations of up to 6%, with theenhancement being directly dependent on the concentration ofthe sugar. The exogenously added sugar was not metabolized andremained unchanged. Sorbitol also promoted the production ofarbutin in a similar manner. Sucrose improved the viability of cells and, in cultures suppliedwith sucrose and HQ, the activity of UDP-glucose: hydroquinoneglucosyltransferase increased over a much longer period of timethan that in control cultures supplemented with HQ only. (Received December 11, 1989; Accepted March 26, 1990)     

Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.     

Impairment in reproductive development is a major factor limiting yield of black gram under zinc deficiency

Black gram [Vigna mungo (L.) Hepper] cv. IPU 94 plants grown in sand culture with deficient zinc (0.1 μM Zn) nutrition and those deprived of normal (1 μM) Zn supply at the initiation of flowering, showed decrease in dry matter production and especially seed yield. These plants showed a decrease in the size of anthers and stigmatic heads, pollen producing capacity of the anthers and stigmatic exudations. Zn deficiency caused structural alterations in exine and retarded germination of pollen grains and tube growth. The pollen extracts and stigmatic exudates of the Zn-deficient plants showed increase in activity of acid phosphatase isoforms and inhibition of esterase isoforms. Zn deficiency led to decrease in number of pods, seeds per pod and seed mass, altered seed coat topography and reduced seeds germinability. Low seed yield under Zn deficiency is attributed to a role of Zn in pollen function, as also in pollen-pistil interaction conducive to fertilization and development of seeds.     

Mutation of a LysR-Type Regulator of Antifungal Activity Results in a Growth Advantage in Stationary Phase Phenotype in Pseudomonas aureofaciens PA147-2

The growth advantage in stationary phase (GASP) phenotype was shown to be present in two mutants lacking the antifungal phenotype (Af⁻ mutants) of Pseudomonas aureofaciens PA147-2. Complementation demonstrated a correlation between GASP and the antifungal defect in one strain but not in the second. Sequence analysis revealed the Af⁻ GASP strain had a mutation in a gene (finR) encoding a LysR-type regulator. Antifungal-minus mutants arose in starved cultures, and those aged cultures had increased fitness. Taken together, the results show that there are at least two paths to the GASP phenotype in P. aureofaciens, one of which results in a concomitant loss of the antifungal phenotype.     

High-level Expression and Purification of a Designed Angiopoietin-1 Chimeric Protein,COMP-Ang1, Produced in Chinese Hamster Ovary Cells

A designed angiopoietin-1 (Ang1) chimeric protein with nonleaky angiogenic activity, COMP-Ang1, is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (>20 mug/mL) of COMP-Ang1 and an amino-terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, 0.32, and 1 muM. The COMP-Ang1 secreted from rCHO cells was purified at a purification yield of 40.3% from the culture medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secrete COMP-Ang1 in homopentameric and homotetrameric glycoprotein forms. Furthermore, COMP-Ang1 binds to the Tie2 receptor and phosphorylates Tie2, indicating its potential for therapeutic angiogenesis.     

Functional Coupling of the NK1 Tachykinin Receptor to Phospholipase D in Chinese Hamster Ovary Cells and Astrocytoma Cells

Abstract: In [³H]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK₁ tachykinin receptor, the selective NK₁ agonist [Pro⁹]substance P ([Pro⁹]SP) time and concentration dependently stimulated the formation of [³H]phosphatidylethanol in the presence of ethanol. This [Pro⁹]SP-induced activation of phospholipase D (PLD) was blocked by NK₁ receptor antagonists and poorly or not mimicked by NK₂ and NK₃ agonists, respectively. In confirmation of previous observations, [Pro⁹]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro⁹]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a G_i/G_o protein is not involved in these different signaling pathways. The activation of PLD by [Pro⁹]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro⁹]SP-evoked response was neither affected by Rp-8-bromoadenosine 3′,5′-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK₁ receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro⁹]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK₁ receptors.