Assembly of vimentin in cultured cells varies with cell type

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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Downregulation of renal TonEBP in hypokalemic rats

AJP Renal Physiology ◽

10.1152/ajprenal.00502.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. F408-F415 ◽

Cited By ~ 22

Author(s):

Un Sil Jeon ◽

Ki-Hwan Han ◽

Soo-Hyun Park ◽

Sang Do Lee ◽

Mee Rie Sheen ◽

...

Keyword(s):

Cultured Cells ◽

Renal Medulla ◽

Distal Tubule ◽

Cell Type ◽

Outer Medulla ◽

Sodium Transporters ◽

Enhancer Binding Protein ◽

Collecting Ducts ◽

Cell Type Specific ◽

Underlying Mechanisms

Hypokalemia causes a significant decrease in the tonicity of the renal medullary interstitium in association with reduced expression of sodium transporters in the distal tubule. We asked whether hypokalemia caused downregulation of the tonicity-responsive enhancer binding protein (TonEBP) transcriptional activator in the renal medulla due to the reduced tonicity. We found that the abundance of TonEBP decreased significantly in the outer and inner medullas of hypokalemic rats. Underlying mechanisms appeared different in the two regions because the abundance of TonEBP mRNA was lower in the outer medulla but unchanged in the inner medulla. Immunohistochemical examination of TonEBP revealed cell type-specific differences. TonEBP expression decreased dramatically in the outer and inner medullary collecting ducts, thick ascending limbs, and interstitial cells. In the descending and ascending thin limbs, TonEBP abundance decreased modestly. In the outer medulla, TonEBP shifted to the cytoplasm in the descending thin limbs. As expected, transcription of aldose reductase, a target of TonEBP, was decreased since the abundance of mRNA and protein was reduced. Downregulation of TonEBP appeared to have also contributed to reduced expression of aquaporin-2 and UT-A urea transporters in the renal medulla. In cultured cells, expression and activity of TonEBP were not affected by reduced potassium concentrations in the medium. These data support the view that medullary tonicity regulates expression and nuclear distribution of TonEBP in the renal medulla in cell type-specific manners.

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Glass beads load macromolecules into living cells

Journal of Cell Science ◽

10.1242/jcs.88.5.669 ◽

1987 ◽

Vol 88 (5) ◽

pp. 669-678

Author(s):

P.L. McNeil ◽

E. Warder

Keyword(s):

Culture Medium ◽

Small Volume ◽

Cultured Cells ◽

Cell Monolayer ◽

Cell Loss ◽

Living Cells ◽

Glass Beads ◽

Cell Type ◽

Large Numbers ◽

Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

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Verification of Non-thermal Effects of 0.3–0.6 THz-Waves on Human Cultured Cells

Photonics ◽

10.3390/photonics6010033 ◽

2019 ◽

Vol 6 (1) ◽

pp. 33 ◽

Cited By ~ 4

Author(s):

Noriko Yaekashiwa ◽

Hisa Yoshida ◽

Sato Otsuki ◽

Shin’ichiro Hayashi ◽

Kodo Kawase

Keyword(s):

Thermal Effects ◽

Cultured Cells ◽

Biological Effects ◽

Wave Exposure ◽

Cell Type ◽

Practical Applications ◽

Thz Wave ◽

Metabolic Activities ◽

Thz Waves ◽

Made In

Recent progress has been made in the development of terahertz (THz) waves for practical applications. Few studies that have assessed the biological effects of THz waves have been reported, and the data currently available regarding the safety of THz waves is inadequate. In this study, the effect of THz wave exposure on two cultured cells was assessed using a widely tunable THz source with a 0.3–0.6 THz frequency range, which can be used and increased in one GHz increments. The THz waves applied to the cultured cells were weak enough such that any thermal effects could be disregarded. The influence of THz wave exposure on both the proliferative and metabolic activities of these cells was investigated, as well as the extent of the thermal stress placed on the cells. In this work, no measurable effect on the proliferative or metabolic activities of either cell type was observed following the exposure to THz waves. No differences in the quantity of cDNA related to heat shock protein 70 was detected in either the sham or exposure group. As such, no differences in cellular activity between cells exposed to THz waves and those not exposed were observed.

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Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells

Biochemical Journal ◽

10.1042/bj3540017 ◽

2001 ◽

Vol 354 (1) ◽

pp. 17-24 ◽

Cited By ~ 48

Author(s):

Anneliese D. RECKLIES ◽

Chantal WHITE ◽

Lee MELCHING ◽

Peter J. ROUGHLEY

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Cultured Cells ◽

Articular Chondrocytes ◽

Synovial Cells ◽

Mrna Levels ◽

Cell Type ◽

Osteosarcoma Cells ◽

Predominant Species ◽

Tgf Β1

Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-β1) and cytokines [interleukin 1β1 (IL-1β)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1β, and showed a unique synergistic response when IL-1β was used in combination with TGF-β1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-β1/IL-1β, the magnitude of the change was far less than the effect on HA synthesis. Our data thus suggest that HAS gene usage is tissue specific, and the regulation by growth factors is unique for each HAS gene and is further modulated by cell-specific factors. In addition, regulation of HA biosynthesis appears to be multi-faceted, with control of HAS gene expression and mRNA levels being only one aspect of this process.

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Cell Type Matters: Competence For Alkaloid Metabolism Differs In Two Seed-Derived Cell Strains of Catharanthus Roseus.

10.21203/rs.3.rs-852281/v1 ◽

2021 ◽

Author(s):

Manish L. Raorane ◽

Christina Manz ◽

Sarah Hildebrandt ◽

Marion Mielke ◽

Marc Thieme ◽

...

Keyword(s):

Plant Cell ◽

Catharanthus Roseus ◽

Cultured Cells ◽

Cell Type ◽

Precursor Feeding ◽

Mature Embryos ◽

Limited Success ◽

Terpene Indole Alkaloids ◽

Cell Strains ◽

Alkaloid Metabolism

Abstract Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question, how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seeds of Catharanthus roseus were found not only to differ morphologically, but also in their metabolic competence. This differential competence became manifest under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not just “de-differentiated”, but can differ in metabolic competence. In addition to elicitation, and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.

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In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

Journal of General Virology ◽

10.1099/vir.0.011650-0 ◽

2009 ◽

Vol 90 (8) ◽

pp. 1943-1950 ◽

Cited By ~ 31

Author(s):

Thierry M. Work ◽

Julie Dagenais ◽

George H. Balazs ◽

Joanne Schumacher ◽

Teresa D. Lewis ◽

...

Keyword(s):

Cultured Cells ◽

Tumour Size ◽

Chelonia Mydas ◽

Tumour Cells ◽

Dermal Fibroblasts ◽

Host Cells ◽

Positive Staining ◽

Cell Type ◽

Green Turtles

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

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Ectopic synthesis of epidermal cytokeratins in pancreatic islet cells of transgenic mice interferes with cytoskeletal order and insulin production.

The Journal of Cell Biology ◽

10.1083/jcb.120.3.743 ◽

1993 ◽

Vol 120 (3) ◽

pp. 743-755 ◽

Cited By ~ 48

Author(s):

M Blessing ◽

U Rüther ◽

W W Franke

Keyword(s):

Transgenic Mice ◽

Beta Cells ◽

Cell Function ◽

Cultured Cells ◽

Ectopic Expression ◽

Epidermal Keratinocytes ◽

Cell Type ◽

Cell Functions ◽

Insulin Promoter ◽

Cell Type Specific

The members of the multigene family of intermediate filament (IF) proteins are expressed in various combinations and amounts that are specific for a given pathway or state of differentiation. Previous experiments in which the cell type-specific IF cytoskeleton was altered by introducing foreign IF proteins into cultured cells or certain tissues of transgenic animals have shown a remarkable tolerance, without detectable interference with cell functions. To examine the importance of the cell type-specific cytokeratin (CK) IF pattern, we have studied the ectopic expression of CK genes in different epithelia of transgenic mice. Here we report changes observed in the beta cells of pancreatic islets expressing the genes for human epidermal CKs 1 and/or 10 brought under control of the rat insulin promoter. Both genes were efficiently expressed, resulting in the appearance of numerous and massive bundles of aggregated IFs, resembling those of epidermal keratinocytes. While the synthesis of epidermal CK 10 was readily accommodated and compatible with cell function, mice expressing CK 1 in their beta cells, alone or in combination with CK 10, developed a special form of diabetes characterized by a drastic reduction of insulin-secretory vesicles and of insulin-and CK 1-producing cells. In many CK 1-producing cells, accumulations of fibrous or granular material containing CK 1 were also seen in the nucleus. This demonstration of functional importance of the specific CK-complement in an epithelial cell indicates a contribution of cell type-specific factors to cytoplasmic IF compartmentalization and that the specific CK complement can be crucial for functions and longevity of a given kind of epithelium.

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The effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-induced Ca2+ mobilization in freshly isolated and cultured mouse lacrimal acinar cells

Biochemical Journal ◽

10.1042/bj3470077 ◽

2000 ◽

Vol 347 (1) ◽

pp. 77-82 ◽

Cited By ~ 9

Author(s):

Peter M. SMITH ◽

Alexander R. HARMER ◽

Andrew J. LETCHER ◽

Robin F. IRVINE

Keyword(s):

Cultured Cells ◽

Physiological Role ◽

Cell Type ◽

Isolated Cells ◽

Calcium Store ◽

Inositol Polyphosphates ◽

Order Of Magnitude ◽

Lacrimal Acinar Cells ◽

Experimental Difference ◽

Stimulation Of

Earlier reports have shown a remarkable synergism between InsP4 and InsP3 [either Ins(1,4,5)P3 or Ins(2,4,5)P3] in activating Ca2+-dependent K+ and Cl- currents in mouse lacrimal cells [Changya, Gallacher, Irvine, Potter and Petersen (1989) J. Membr. Biol. 109, 85-93; Smith (1992) Biochem. J. 283, 27-30]. However, Bird, Rossier, Hughes, Shears, Armstrong and Putney [(1991) Nature (London) 352, 162-165] reported that they could see no such synergism in the same cell type. A major experimental difference between the two laboratories lies in whether or not the cells were maintained in primary culture before use. Here we have compared directly the responses to inositol polyphosphates in freshly isolated cells versus cells cultured for 6-72 h. In the cultured cells, Ins(2,4,5)P3 at 100 μM produced a robust stimulation of K+ and Cl- currents, as much as an order of magnitude greater than that observed in the freshly isolated cells. However, the freshly isolated cells could be restored to a sensitivity similar to cultured cells by the addition of InsP4 at a concentration two orders of magnitude lower than that of Ins(2,4,5)P3. We discuss the implications of this with respect to the actions of InsP4, including the possibility that disruption of the cellular structure during the isolation of the cells exposes an extreme manifestation of a possible physiological role for InsP4 in controlling calcium-store integrity.

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Tradeoffs for a viral mutant with enhanced replication speed

10.1101/2021.03.24.436823 ◽

2021 ◽

Author(s):

Matthew R. Lanahan ◽

Julie K. Pfeiffer

Keyword(s):

Viral Replication ◽

Cultured Cells ◽

Wild Type Virus ◽

Specific Cell ◽

Single Mutation ◽

Cell Type ◽

Viral Mutant ◽

Capsid Protein Vp1 ◽

Capsid Stability

AbstractRNA viruses exist as genetically heterogeneous populations due to high mutation rates and many of these mutations reduce fitness and/or replication speed. However, it is unknown whether mutations can increase replication speed of a virus already well adapted to replication in cultured cells. By sequentially passaging coxsackievirus B3 in cultured cells and collecting the very earliest progeny, we selected for increased replication speed. We found that a single mutation in a viral capsid protein, VP1-F106L, was sufficient for the fast-replication phenotype. Characterization of this mutant revealed quicker genome release during entry compared to wild-type virus, highlighting a previously unappreciated infection barrier. However, this mutation also reduced capsid stability in vitro and reduced replication and pathogenesis in mice. These results reveal a tradeoff between overall replication speed and fitness. Importantly, this approach— selecting for the earliest viral progeny—could be applied to a variety of viral systems and has the potential to reveal unanticipated inefficiencies in viral replication cycles.SignificanceViruses have characteristic replication speeds within a given cell type. Many factors can slow the rate of viral replication, including attenuating mutations and host antiviral responses. However, it has been unclear whether it would be possible to “speed up” a virus that already replicates efficiently in a specific cell type. Here, we selected for a mutant coxsackievirus with enhanced replication speed by sequentially harvesting the very earliest progeny in multiple rounds of selection. A single mutation conferred the fast replication phenotype. While this mutant virus has enhanced replication in cultured cells due to faster genome uncoating, it was attenuated in mice. These results highlight selective pressures that shape viral populations in different environments.

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