THE USE OF FARADAY INSTABILITY TO PRODUCE DEFINED TOPOLOGICAL ORGANIZATION IN CULTURES OF MAMMALIAN CELLS

2002 ◽  
Vol 12 (09) ◽  
pp. 2009-2019 ◽  
Author(s):  
SEIJI TAKAGI ◽  
VALENTIN KRINSKY ◽  
ALAIN PUMIR ◽  
CHRISTIAN FRELIN
Keyword(s):  
Pattern Formation ◽  
Time Scale ◽  
Mammalian Cells ◽  
Prolonged Exposure ◽  
Time Window ◽  
Cell Attachment ◽  
Hydrodynamic Instability ◽  
Cell Types ◽  
Faraday Instability

Pattern formation occurs spontaneously in endothelial cell cultures, leading to the formation of capillary networks, which eventually grow to form blood vessels. This phenomenon occurs on a time scale of a few days. We show here that patterns can also be induced on a much shorter time scale, by using the Faraday hydrodynamic instability, resulting from an oscillatory motion of the container. Close to the threshold of instability, the patterns observed are very sharp concentric rings or stripes. The patterns can be induced only inside a very narrow time window, ~ 5 min. Cells attachment then develops, and pattern formation can no longer be induced. The time window for pattern formation was diminished by favoring cell attachment, for instance by treating culture dishes with cationic macromolecules, such as poly-L-Lysine. It was increased by cooling the cells to 18°C, or by a prolonged exposure of the cells to trypsin, which is known to digest adhesion molecules. The Faraday instability leads to a method to characterize cell attachment. It also permits the production of heterogeneous cultures with several cell types, with a well controlled heterogeneity. This can be used to study heterotypic cell interactions in vitro.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

scholarly journals Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

2003 ◽  
Vol 2003 (2) ◽  
pp. 79-91 ◽  
Author(s):  
Lindsay J. Stanbridge ◽  
Vincent Dussupt ◽  
Norman J. Maitland
Keyword(s):  
Prostate Cancer ◽  
Gene Therapy ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Primary Tumour ◽  
Genetic Material ◽  
Cell Types ◽  
Attractive Alternative

Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

EFFECTS OF ANAEROBIOSIS ON THE RATES OF MULTIPLICATION OF MAMMALIAN CELLS CULTURED IN VITRO

1960 ◽  
Vol 38 (1) ◽  
pp. 871-878 ◽  
Author(s):  
Samuel Dales
Keyword(s):  
Mammalian Cells ◽  
Glucose Utilization ◽  
Lactic Acid Production ◽  
Cell Types ◽  
In Vitro Cultures ◽  
Cell Multiplication ◽  
Embryonic Mouse ◽  
Mouse Cells ◽  
Oxidation Of Glucose

To test the effects of anaerobiosis on the rate of multiplication and carbohydrate metabolism of mammalian cells in vitro, cultures of a 'permanent' line, Earle's L strain cells, and of freshly explanted embryonic mouse cells were propagated in the presence and absence of oxygen. Contrary to the findings of several other investigators, our results show that the multiplication of both cell types was depressed by anaerobiosis. Anaerobiosis for at least 7 days, did not, however, bring about unbalanced growth in L cells, nor did it affect their capability to divide rapidly soon after they were returned to aerobic conditions. From the rates of glucose utilization, lactic acid production, and cell multiplication it was estimated that the rate of division in the two cell types studied was proportional to the energy which could be released from either glycolysis or complete oxidation of glucose.

Nanoscale RGD Peptide Organization Regulates Cell Proliferation and Differentiation

2004 ◽  
Vol 845 ◽  
Author(s):  
Susan Hsiong ◽  
Kuen Yong Lee ◽  
Eben Alsberg ◽  
David Mooney
Keyword(s):  
Cell Attachment ◽  
Cell Types ◽  
Important Variable ◽  
Rgd Peptide ◽  
Polymer Chains ◽  
Cell Phenotype ◽  
Rgd Peptides ◽  
Ligand Density ◽  
Proliferation And Differentiation

ABSTRACTRGD (arginine-glycine-aspartic acid) containing peptide sequences, common cell attachment sites present in many extracellular matrix (ECM) proteins, mediate many important cellular processes. The role of nanoscale organization of RGD peptides in the regulation of the adhesion, proliferation and differentiation of both preosteoblasts (MC3T3-E1) and multipotential (D1) cell lines in vitro was investigated in this study. Alginate polymer chains with varying RGD peptide degree of substitution were mixed with unmodified polymer chains at different ratios to allow variation of RGD peptide spacing in the nanometer scale, independently of the overall bulk density of peptides presented from the material. Proliferation of both cell types was observed to be closely correlated to RGD island (defined as a cluster of RGD peptides) spacing, independently of overall bulk ligand density, following cell adhesion to alginate hydrogels. Increased RGD island spacing was observed to promote spreading of MC3T3-E1 cells while simultaneously suppressing their proliferation. However, increased RGD island spacing decreased spreading of D1 cells while also decreasing proliferation. Moreover, differentiation of preosteoblasts was significantly upregulated in response to decreased RGD island spacing, whereas differentiation of multipotential cells was modestly regulated by this variable. These results demonstrate that the nanoscale organization of adhesion ligands may be an important variable in controlling cell phenotype and function. In addition, cellular responses to nanoscale ligand organization differ depending on the cell type, and this may be related to the differentiation stage of the cells.

scholarly journals Polarization of fluorescein fluorescence in single cells.

1979 ◽  
Vol 27 (1) ◽  
pp. 49-55 ◽  
Author(s):  
R Udkoff ◽  
A Norman
Keyword(s):  
Mammalian Cells ◽  
Single Cells ◽  
Classical Model ◽  
Cell Types ◽  
Wavelength Shift ◽  
Two Peaks ◽  
Vitro Data ◽  
P Distribution ◽  
In Vitro Data

Measurement of fluorescence polarization (P) gives information about the immediate environment of the fluorescent molecule. We used a flow polarimeter to investigate the factors influencing P of fluorescein in mammalian cells to determine whether such measurements are useful for characterizing heterogeneous cell populations. Fluorescein was introduced into cells by incubation with FDA. Measurements of the intensity of fluorescence (TI) and polarization (P) revealed an unexpected dependence: P decreased with increasing intensity of fluorescence. This may be accounted for by the classical model of the binding of small molecules to protein in which P is dependent on the ratio bound to unbound molecules. We have been able to estimate the quenching due to binding and construct a Scatchard plot. We estimated a wavelength shift from in vitro data consistent with the dependence of P on wavelength seen in our cell work. Generally, the distributions of P are symmetrical. Photon statistics broadens the P distribution of dim cells. However, structure does develop in the P distribution when the cells are deprived of calcium or incubated in the cold. This appears as a shoulder on the P distribution or resolves into two peaks. Calcium deprivation may differentially affect a subpopulation of cells whose significance remains to be explored in various cell types.

Uptake of [3H]Uridine into Precursor Pools and RNA in Osteogenic Cells

1967 ◽  
Vol 2 (1) ◽  
pp. 39-56
Author(s):  
MAUREEN OWEN
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Rna Synthesis ◽  
Degradation Products ◽  
Cell Types ◽  
Water Soluble ◽  
Radioactive Label ◽  
Cytoplasmic Rna ◽  
Nuclear Rna

Young rabbits were given a single intraperitoneal injection of [3H]uridine. Using the technique of water-soluble autoradiography a study was made of the uptake of the radioactive label into soluble precursors and RNA in cells on an actively growing bone surface. Labelling of the soluble intracellular pools was immediate, but incorporation of label from these pools into RNA was not completed until 24 h after injection. At this time all the label in the sections was in RNA but this represented only 30% of the total label initially in the soluble pools. This means that 70% of the label is lost from the cell in the first 24 h either as degradation products of RNA synthesis or by other as yet unknown mechanisms. The pattern of labelling of the RNA was similar to that previously found for other mammalian cells in vivo or in vitro. There was a rapid uptake of label into nuclear RNA which reached a maximum by 2 h after injection and a slower uptake into cytoplasmic RNA which reached a maximum by 24 h after injection. There was a slow loss of label from the cells after 24 h indicating a half-life of about 8 days for this relatively stable RNA. A comparison was made of RNA synthesis in the proliferating preosteoblasts and the highly differentiated non-dividing osteoblasts. Labelling of the nuclear RNA for the two cell types was identical. The rate of labelling of the cytoplasmic RNA was similar for the two cell types but the maximum level of labelling in the cytoplasm of the osteoblasts was 2 to 3 times that in the preosteoblasts. This could be correlated with the more active protein synthesis by the osteoblasts. There was a slow loss of labelled RNA by the osteoblasts and preosteoblasts and a rapid loss by the osteocytes after the cells had been incorporated within the bone. It was suggested that this loss paralleled the decline in the rate of protein synthesis by the cells as their environment changed.

scholarly journals Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation

2006 ◽  
Vol 87 (8) ◽  
pp. 2263-2268 ◽  
Author(s):  
Dennis J. Pierro ◽  
Ma Isabel Salazar ◽  
Barry J. Beaty ◽  
Ken E. Olson
Keyword(s):  
Cell Culture ◽  
Dengue Virus ◽  
Virus Type ◽  
Mammalian Cells ◽  
Infectious Clone ◽  
Cell Types ◽  
Dengue Virus Type 2

A full-length infectious cDNA clone (ic) was constructed from the genome of the dengue virus type 2 (DENV-2) Jamaica83 1409 strain, pBAC1409ic, by using a bacterial artifical chromosome plasmid system. Infectious virus was generated and characterized for growth in cell culture and for infection in Aedes aegypti mosquitoes. During construction, an isoleucine to methionine (Ile→Met) change was found at position 6 in the envelope glycoprotein sequence between low- and high-passage DENV-2 1409 strains. In vitro-transcribed genomic RNA of 1409ic with E6-Ile produced infectious virions following electroporation in mosquito cells, but not mammalian cells, while 1409ic RNA with an E6-Met mutation produced virus in both cell types. Moreover, DENV-2 1409 with the E6-Ile residue produced syncytia in C6/36 cell culture, whereas viruses with E6-Met did not. However, in vitro cell culture-derived growth-curve data and in vivo mosquito-infection rates revealed that none of the analysed DENV-2 strains differed from each other.

scholarly journals TAPping into the treasures of tubulin using novel protein production methods

2018 ◽  
Vol 62 (6) ◽  
pp. 781-792
Author(s):  
Nuo Yu ◽  
Niels Galjart
Keyword(s):  
Mammalian Cells ◽  
Gene Families ◽  
Cell Types ◽  
Cellular Functions ◽  
Tubulin Isotypes ◽  
Associated Proteins ◽  
Cytoskeletal Elements ◽  
Different Cell Types ◽  
Β Tubulin

Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the ‘tubulome’ of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types.

scholarly journals A novel physical and functional association between nucleoside diphosphate kinase A and AMP-activated protein kinase α1 in liver and lung

2005 ◽  
Vol 392 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Russell M. Crawford ◽  
Kate J. Treharne ◽  
O. Giles Best ◽  
Richmond Muimo ◽  
Claudia E. Riemen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Nucleoside Diphosphate Kinase ◽  
Cell Types ◽  
Acid Synthesis ◽  
Cellular Fatty Acid ◽  
Energy Status ◽  
Amp Activated Protein Kinase

Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (∼16–20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK α1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the α1 (but not α2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK α1-mediated phosphorylation is present in mammalian cells.

Injection Molding of ProNectin®F Dispersed in Polystyrene for The Fabrication of Plastic Ware Activated Towards Attachment of Mammalian Cells

1993 ◽  
Vol 330 ◽  
Author(s):  
Erwin R. Stedronsky ◽  
Joseph Cappello ◽  
Samuel David ◽  
David M. Donofrio ◽  
Tina Mcarthur ◽  
...  
Keyword(s):  
Injection Molding ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
De Novo ◽  
Cell Attachment ◽  
Biologically Active ◽  
Polymeric Surfactant ◽  
New Paradigm

ABSTRACTProNectin®F is a recombinant engineered protein polymer of de novo design which incorporates the RGD epitope recognized by mammalian cell integrins. It is biologically active as a cell attachment protein, manifests properties of a planar polymeric surfactant, and is extremely resistant to thermal degradation. ProNectin®F was dispersed onto polystyrene powder, fabricated into plastic ware through injection molding, and the plastic ware was shown to have cell attachment activity. This technology represents a new paradigm for the production of plastic ware useful for mammalian cell culture under serum free conditions; and more generally, for the production of molded devices for use in contact with cells in vitro or in vivo.

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