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1.
Carinhas N Bernal V Yokomizo AY Carrondo MJ Oliveira R Alves PM 《Applied microbiology and biotechnology》2009,81(6):1041-1049
One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant
proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand
this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus
expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities
of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used,
with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using
a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific
yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers
as high as 2.6 × 1010 IP.mL−1 were obtained in cultures infected at 3.5 × 106 cells.mL−1, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange. 相似文献
2.
Recombinant adenoviral vectors (AdV) have proven to be highly efficient for the delivery and expression of foreign genes in a broad spectrum of cell types and species both for vaccination and gene therapy in a number of specific applications. In this study, the effect of ammonia production on intracellular pH (pH(i)) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pH(i) was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3x10(6)cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pH(i) in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pH(i) effect upon AdV production, 293 cells were infected at a CCI of 1 x 10(6)cell/ml in the non-ammoniagenic medium with a manipulated pH(i) as observed at the time of infection at CCI of 3 x 10(6)cell/ml in the ammoniagenic (pH(i) 7.0) and non-ammoniagenic (pH(i) 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pH(i). Thus, the importance of controlling all the factors contributing to pH(i) on AdV production, such as ammonia production, has been established. 相似文献
3.
Yung-Shyeng Tsao Russell Condon Eugene Schaefer Peggy Lio Zhong Liu 《Cytotechnology》2001,37(3):189-198
Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum
weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation
reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing
medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in
shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed
by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation
resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate,
N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production
in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other
E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
4.
Calcium-dipicolinate (Ca-DPA)-rich and Ca-DPA-deficientBacillus cereus spores were incubated in a synthetic medium with germination stimulants and in bactopeptone medium with a fairly high calcium
ion concentration. In the complex medium the germination of Ca-DPA-rich spores was completely blocked at a concentration of
0.5m CaCl2, whereas the complete blockage of germination in the synthetic medium required higher concentrations (0.6–0.8m) of calcium chloride. Ca-DPA-deficient spores germinated more slowly and less completely in the synthetic medium than in
the bactopeptone medium. The germination of these spores took place, however, even at higher calcium ion concentrations (0.6–0.8m). On the contrary, lower calcium chloride concentrations (0.1–0.4m) accelerated the germination of these spores in the synthetic medium and the final percentage of phase-dark and stainable
spores was higher. “H-forms” of the Ca-DPA-rich and Ca-DPA-deficient spores prepared by acid titration germinated in both
media. The germination of the latter spores being slower and proceeding less completely. “H-forms” germinated completely or
partially in media with a high concentration of calcium chloride. The percentage of germinated spores, however, was strongly
influenced by the concentration of this cation, especially the “H-forms” of Ca-DPA-deficient spores. Moreover, the germination
of Ca-DPA-deficient spores in this medium was affected by the length of previous storage and, in the case of “H-forms” by
the pH at which they were titrated. It was assumed that the increased permeability of calcium into the calciumundersaturated
spore periphery in Ca-DPA-deficient and in “H-forms” of spores of both types co-determines (in the presence of germinants)
the germinability of bacterial spores. 相似文献
5.
Jianyong Wu Glenn A. King Andrew J. Daugulis Peter Faulkner Mattheus F. A. Goosen 《Cytotechnology》1992,9(1-3):141-147
Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the
gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and
Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus
and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually
the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI
(multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both
cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant
temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little
or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor)
serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase).
A cell density as high as 1×107 cells.ml−1, and a β-gal concentration of up to 104,000 unit.ml−1 were achieved. 相似文献
6.
Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate
interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use
of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be
fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process
using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of
homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3
ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide
mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene
plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and
such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM
concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between
N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using
BAP conjugates of glycosyl epitopes. 相似文献
7.
Adenoviruses are promising vectors for gene therapy. The production of adenoviral vectors (AdV), however, is limited by the
cell density effect, namely when cell infection is performed at above 106 cells/ml, a drop in cell-specific adenovirus productivity occurs. Our results also show that the coxsackie adenovirus receptor
(CAR) plays an important role in AdV production. CAR expression of infected cells varied with culture time and the cell-specific
AdV productivity dropped rapidly along with decreased CAR expression. Furthermore, CAR expression of cells was maintained
at a high level by replacing the medium or supplementing it with trichostatin A, which could improve the cell-specific productivity.
Thus, a higher CAR expression level at infection time could improve cell-specific AdV productivity at high cell densities. 相似文献
8.
Wang X Wang X Yin M Xiao Z Ma C Lin Z Wang PG Xu P 《Applied microbiology and biotechnology》2007,76(2):321-328
Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from
orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst
in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells
exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in
the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were
performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH2PO4 and K2HPO4), MgCl2, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating
that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization
by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l−1) UMP was accumulated in 24 h from 38.5 mM (6 g l−1) orotic acid. The yield was threefold higher than the original UMP yield before optimization. 相似文献
9.
Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues 总被引:1,自引:0,他引:1
Valeria M. Rodas Fabiano H. Marques Marcelo T. Honda Daniela M. Soares Soraia A. C. Jorge Marta M. Antoniazzi Claudia Medugno Maria E. B. Castro Bergmann M. Ribeiro Marlinda L. Souza Aldo Tonso Carlos A. Pereira 《Cytotechnology》2005,48(1-3):27-39
We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors.
We have assayed the kLa of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients
consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than
in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs
production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a
cell concentration at infection (CCI) of 1 to 2.5×106 cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed
to be dependent on the MOI used, and the μX at the exponential growth phase in infected and non-infected cultures were, respectively,
of 0.2832 and 0.3914 (day−1). The glucose consumption and lactate production were higher in the infected cultures (μGlucose and μLactate of, respectively,
0.0248 and 0.0089×10−8 g/cell×day in infected cultures and 0.0151 and 0.0046×10−8 g/cell×day in non infected ones). The glutamine consumption did not differ in both cultures (μGlutamine of 0.0034 and 0.0037×10−8 g/cell×day in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a
higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these
cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective
decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance
of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 106 to 2×106 cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high
MOI showed comparable DL50. Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better
environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB
virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase
in the DL50. The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered
DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy
(TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described
here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control. 相似文献
10.
D. Gaillard R. Négrel G. Serrero-Davé C. Cermolacce G. Ailhaud 《In vitro cellular & developmental biology. Plant》1984,20(2):79-88
Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike
cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin,
transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium
is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet
extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other
established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular
fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these
cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert
to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition
of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements
for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.
This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant
4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la
Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006). 相似文献
11.
Two Different Serum-free Media and Osmolality Effect Upon Human 293 Cell Growth and Adenovirus Production 总被引:1,自引:0,他引:1
Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus
are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted
purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called
‘cell density effect’, i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection.
Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus
production at cell densities higher than 1×106 cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when
the infection was performed at 1×106 cells/ml, at 3×106 cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration
at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies,
which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus
productivity was only affected for osmolalities higher than 430 mOsm.
Revisions requested 24 August 2005; Revisions received 9 September 2005 相似文献
12.
Kamilla Swiech Nickeli Rossi Bruna Gabriela Silva Soraia A. C. Jorge Renato Mancini Astray Cláudio Alberto Torres Suazo 《Cytotechnology》2008,57(1):61-66
Although several reports have been published on recombinant protein expression using Drosophila cells, information on their metabolism and growth in vitro is relatively scarce. In the present study, we have analyzed the
growth and metabolism of transfected S2 cells (S2AcRVGP) in bioreactor cultures with serum-free medium Sf900 II, to evaluate
its potential for mass production of a rabies virus glycoprotein (RVGP). Cells were cultured in a 3 l-stirred-tank bioreactor
at 28 °C with pH controlled at 6.2 and dissolved oxygen at 50% air saturation. The cells attained a specific growth rate and
maximum cell density as high as 0.084 h−1 and 2.3 × 107 cell ml−1, respectively. The main substrates consumed during this rapid growth phase were glucose, glutamine and proline. An atypical
accumulation of ammonia and alanine was observed in the culture medium, up to 62 mM and 47 mM, respectively, but lactate was
produced in low levels. After exhaustion of glutamine and proline as energy sources, alanine was consumed and production of
ammonia increased. The production of recombinant RVGP reached concentrations as high as 178 μg l−1. Premature exhaustion of glutamine, serine and cysteine could be related to degradation of the recombinant glycoprotein.
In general, the results demonstrated that S2AcRVGP can be considered an effective vehicle for large-scale recombinant glycoprotein
expression and that several critical factors of the bioprocess could be optimized to increase the quality and productivity
of the RVGP. 相似文献
13.
A mathematical model of HIV dynamics in the presence of a rescuing virus with replication deficiency
Recently, an enzyme (Cre recombinase) has been developed by directed evolution that successfully removes the HIV genome from
the nuclear DNA of infected cells. To explore this idea further, we hypothesized that a replication deficient virus (called
“police virus”), added externally, can deliver such a recombinase which excises the integrated HIV DNA from the genome of
infected cells. Such a “police virus” could attack and remove the integrated provirus which is not possible using contemporary
strategies. The hypothesis was tested by developing a mathematical model that describes the dynamics of virus-host cell interaction
and the consequences of introducing the “police virus”. The simulations show that such a therapeutic vector may eradicate
all HIV viruses from the system in the long term. All components of the HIV infection (free virus, latently, and actively
infected cells) can be cleared and the system ends up only with susceptible CD4+ cells. The proposed model may provide new
insights in the dynamical behavior and future alternative treatments of HIV. 相似文献
14.
Yan Xu Haihui Ye Jun Ma Huiyang Huang Guizhong Wang 《In vitro cellular & developmental biology. Animal》2010,46(8):708-717
Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in
modified Medium 199 (M199) medium than that in Liebowitz’s L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph
of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by
initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics
were identified in the neuron outgrowth. They were “small cells”, “veilers”, “branchers”, “multipolar cells”, “super-large
cell”, and “bipolar cells”. Among all of the cells, bipolar cells were identified for the first time in crustacean neurons
culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual
degradation. 相似文献
15.
Jeffrey W. Adelberg Maria P. Delgado Jeffery T. Tomkins 《In vitro cellular & developmental biology. Plant》2010,46(1):95-107
Residual nutrients from Murashige and Skoog medium were analyzed following a 5-wk multifactor experiment. Plant density, sugar
concentration, and plant growth regulators (benzyladenine and ancymidol) were examined using four genotypes of daylily (Hemerocallis) to determine which factors most influenced nutrient use. Active nutrient uptake was observed for 11 nutrients (potassium,
sodium, copper, phosphorus, iron, calcium, magnesium, manganese, boron, sulfur, and zinc) with lower concentrations in spent
medium than in the tissue water volume (fresh-dry mass expressed as mL H2O). Two patterns of nutrient use were visualized by correlative analysis of nutrient uptake. Greatest growth lowered plant
nutrient concentrations of potassium, sodium, phosphorus, iron, and copper in all genotypes, and luxuriant uptake was indicated
with least growth. Potassium, sodium, iron, and copper concentrations in plant dry matter were equal to or exceeded what is
observed in vigorously growing nursery plants. However, phosphorus concentration in plant dry matter was low enough to be
considered deficient when compared to Hemerocallis plants in nursery production. With a second group of nutrients (calcium, magnesium, manganese, and boron), the genotype,
“Barbara Mitchell” lacked active uptake and was deficient. Calcium concentration was low in all plants compared to Hemerocallis grown under nursery conditions (“Barbara Mitchell” was the lowest concentration) despite active uptake by the other three
genotypes—“Brocaded Gown,” “Mary’s Gold,” and “Heart of a Missionary.” Magnesium concentration in these three genotypes was
low enough in vessels with greatest growth to question its adequacy at high densities. Increased sucrose in medium reduced
the dry matter concentrations of all tested nutrients. Plant growth regulators had less impact on nutrient use than genotype
and plant density. Nutrient uptake may be an important physiological component of genotypic variation. 相似文献
16.
Van B. Lu S. Balasubramanyan J. E. Biggs M. J. Stebbing S. L. Gustafson K. Todd A. Lai D. Dawbarn W. F. Colmers K. Ballanyi P. A. Smith 《Neurophysiology》2007,39(4-5):272-283
Chronic constriction injury (CCI) of the rat sciatic nerve increases the dorsal horn excitability. This “central sensitization”
leads to behavioral manifestations analogous to those related to human neuropathic pain. We found, using whole-cell recording
from acutely isolated spinal cord slices, that 7-to 10-day-long CCI increases excitatory synaptic drive to putative excitatory
“delay”-firing neurons in the substantia gelatinosa but attenuates that to putative inhibitory “tonic”-firing neurons. A defined-medium organotypic culture (DMOTC) system was
used to investigate the long-term actions of brain-derived neurotrophic factor (BDNF) as a possible instigator of these changes.
When all five neuronal types found in the substantia gelatinosa were considered, BDNF and CCI produced similar patterns, or “footprints,” of changes across the whole population. This pattern
was not seen with another putative “pain mediator,” interleukin 1β. Thus, BDNF decreased synaptic drive to “tonic” neurons
and increased synaptic drive to “delay” neurons. Actions of BDNF on “delay” neurons were presynaptic and involved increased
mEPSC frequency and amplitude without changes in the function of postsynaptic AMPA receptors. By contrast, BDNF exerted both
pre-and post-synaptic actions on “ tonic” cells to reduce the mEPSC frequency and amplitude. These differential actions of
BDNF on excitatory and inhibitory neurons contributed to a global increase in the dorsal horn network excitability as assessed
by the amplitude of depolarization-induced increases in the intracellular [Ca2+]. Experiments with the BDNF-binding protein TrkB-d5 provided additional evidence for BDNF as a harbinger of neuropathic pain.
Thus, the cellular processes altered by BDNF likely contribute to “central sensitization” and hence to the onset of neuropathic
pain.
Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 315–326, July–October, 2007. 相似文献
17.
Stephen D. Bird Robert J. Walker Michael J. Hubbard 《In vitro cellular & developmental biology. Animal》1994,30(7):420-424
Summary The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line,
LLC-PK1, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured
for 3 to 7 wk with (“plus”) and without (“minus”) antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus
cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture
(>20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies
and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties
of LLC-PK1 cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug
effects and raises the general possibility of similar effects on other cultured cells. 相似文献
18.
Susumu Kimura 《Microbiology and immunology》1996,40(3):243-246
Herpes simplex virus type 1 (HSV-1) was reactivated more rapidly in cells of latently infected mouse trigeminal ganglia which were cultured in serum-free medium (after 3.7 days of cultivation) than in those cultured in serum-containing Dulbecco's modified Eagle's medium (after 8.5 days of cultivation). The concentration of calcium ion (Ca2+) in the medium affected HSV-1 reactivation in ganglionic cultures, and 0.9 mM was the optimum concentration for the reactivation. Reactivation was delayed significantly in ganglia put into culture 4 months or more after infection compared with those cultured 1 month after infection. 相似文献
19.
Protein evolution is not a random process. Views which attribute randomness to molecular change, deleterious nature to single-gene
mutations, insufficient geological time, or population size for molecular improvements to occur, or invoke “design creationism”
to account for complexity in molecular structures and biological processes, are unfounded. Scientific evidence suggests that
natural selection tinkers with molecular improvements by retaining adaptive peptide sequence. We used slot-machine probabilities
and ion channels to show biological directionality on molecular change. Because ion channels reside in the lipid bilayer of
cell membranes, their residue location must be in balance with the membrane’s hydrophobic/philic nature; a selective “pore”
for ion passage is located within the hydrophobic region. We contrasted the random generation of DNA sequence for KcsA, a
bacterial two-transmembrane-domain (2TM) potassium channel, from Streptomyces lividans, with an under-selection scenario, the “jackprot,” which predicted much faster evolution than by chance. We wrote a computer
program in JAVA APPLET version 1.0 and designed an online interface, The Jackprot Simulation
, to model a numerical interaction between mutation rate and natural selection during a scenario of polypeptide evolution.
Winning the “jackprot,” or highest-fitness complete-peptide sequence, required cumulative smaller “wins” (rewarded by selection)
at the first, second, and third positions in each of the 161 KcsA codons (“jackdons” that led to “jackacids” that led to the
“jackprot”). The “jackprot” is a didactic tool to demonstrate how mutation rate coupled with natural selection suffices to
explain the evolution of specialized proteins, such as the complex six-transmembrane (6TM) domain potassium, sodium, or calcium
channels. Ancestral DNA sequences coding for 2TM-like proteins underwent nucleotide “edition” and gene duplications to generate
the 6TMs. Ion channels are essential to the physiology of neurons, ganglia, and brains, and were crucial to the evolutionary
advent of consciousness. The Jackprot Simulation illustrates in a computer model that evolution is not and cannot be a random
process as conceived by design creationists. 相似文献
20.
A method was developed for production of a freeze-dried Western equine encephalomyelitis vaccine from virus propagated in chick embryo cell culture monolayers maintained with a serum-free medium. A sufficient concentration of virus accumulated in the cell culture fluids prior to the occurrence of viral cytopathology to permit the production of a vaccine relatively free from serum and cellular proteins. Inoculation with two mouse ld50 doses of virus per 100 tissue culture cells was found to yield reproducible high virus titers at a convenient harvest time. These harvests were inactivated at 22 C by 0.05% formalin within 48 hr. Potency test results, as measured by the protection of immunized guinea pigs against an intracerebral virus challenge, indicated that the vaccine produced from the virus propagated in cell culture was equal in potency to a lot of whole chick embryo vaccine used to immunize laboratory and field workers subject to a high risk of infection. 相似文献