Isolation and characterization of an avian myogenic cell line

Myogenic cell lines have proven extremely valuable for studying myogenesis in vitro. Although a number of mammalian muscle cell lines have been isolated, attempts to produce cell lines from other classes of animals have met with only limited success. We report here the isolation and characterization of seven avian myogenic cell lines (QM1-4 and QM6-8), derived from the quail fibrosarcoma cell line QT6. A differentiation incompetent QM cell derivative was also isolated (QM5DI). The major features of QM cell differentiation in vitro closely resemble those of their mammalian counterparts. Mononucleated QM cells replicate in medium containing high concentrations of serum components. Upon switching to medium containing low serum components, cells withdraw from the cell cycle and fuse to form elongated multinucleated myotubes. Cultures typically obtain fusion indices of 43-49%. Northern blot and immunoblot analyses demonstrate that each differentiated QM cell line expresses a wide variety of genes encoding muscle specific proteins: desmin, cardiac troponin T, skeletal troponin T, cardiac troponin C, skeletal troponin I, alpha-tropomyosin, muscle creatine kinase, myosin light chain 2, and a ventricular isoform of myosin heavy chain. While all QM lines analyzed to date express at least some myosin light chain 2, only one line, QM7, expresses this gene at high levels. Surprisingly, none of the QM lines reported here express any known form of alpha-actin. The absence of sarcomeric actin expression may explain the absence of myofibrils in QM myotubes. These novel features of muscle gene expression in QM cells may prove useful for studying the role of specific muscle proteins during myogenesis. More importantly, however, the isolation of QM cell lines indicates that it may be feasible to isolate other avian myogenic cell lines with general utility for the study of muscle development.     

Isolation and characterization of an adriamycin-resistant breast tumor cell line

Summary An adriamycin-resistant human breast tumor cell line MDA-A1 ^R was generated by step-wise selection in increasing concentrations of drug from the parent cell line MDA-MB-231. MDA-A1 ^R cells grow as loosely attached cell aggregates with a doubling time of 28–32 h; the MDA-MB-231 parent cell line grows as a standard monolayer culture with a 20-h doubling time. The MDA-A1 ^R cell line is highly resistant to adriamycin compared to the parent cell line, and is cross-resistant to velban and colchicine suggestive of a multidrug resistance (MDR) phenotype. MDA-A1 ^R cells exhibit reduced net adriamycin conent as compared to the parent cell line. The MDR-associated P-glycoprotein gene is amplified approximately 10-to 30-fold in MDA-A1 ^R cells. P-glycoprotein sequences are overexpressed in the resistant cells and are stable for up to 13 wk after drug removal. Moreover, MDA-A1 ^R cells show the presence of very high levels of P-glycoprotein. MDA-A1 ^R is thus an in vitro model system to study the mechanism of MDR in human breast cancer. This work was supported in part by grant C30195 from the National Institute of health, Bethesda, MD. Portion of this study appeared as a poster presentation at the Tissue Culture Association meeting, Las Vegas, 1988.     

A highly lipophilic form of aldosterone. Isolation and characterization of an aldosterone dimer

A highly lipophilic form of aldosterone obtained both from incubations of 18-hydroxycorticosterone with quartered rat adrenals and by treatment of aldosterone with acid, was identified as an aldosterone dimer based on its 1H, 13C NMR and mass spectra.     

Isolation and characterization of the erythroid progenitor cell: CFU-E

Erythroid progenitor cells, CFU-E (colony-forming-unit-erythroid), were isolated to practical homogeneity by a combination of three enrichment procedures. CFU-E were generated in large amounts in spleens of mice previously bled and treated with the erythropoiesis-suppressing drug thiamphenicol. The average CFU-E concentration in spleens from mice 4 d after the thiamphenicol-treatment was 10%. These CFU-E were separated from lymphocytes, erythrocytes, and granulocytes and their progenitor cells by centrifugal elutriation and Percoll density gradient centrifugation. A three- to five-fold enrichment was obtained by elutriation, leading to a CFU-E concentration of 45%. With the Percoll gradient another twofold enrichment was achieved, providing us with a 80-100% CFU-E cell population. The overall recovery of CFU-E was 60- 70%. This is a cheap, rapid, and highly efficient method of obtaining large quantities of viable CFU-E. The sequential formation of two-, four-, and eight-cell colonies from CFU-E cultured in vitro was studied. These cells enable us to study the biochemical changes occurring in the differentiation process of an erythroid progenitor cell induced by the hormone erythropoietin. The morphological and some physical and biological properties of these cells are presented.     

Ability of locally irradiated bone marrow to form erythroid colonies in diffusion chambers

It was shown that mouse bone marrow locally exposed to ionizing radiation during the period of T-lymphocytes accumulation can form, int the diffusion chambers in the absence of exogenous erythropoietin, a large number of giant and small erythroid colonies.     

Isolation and characterization of aDatura innoxia cell line resistant to ethanol

A cell line ofDatura innoxia was selected in suspension culture to be resistant to 1% (vol/vol) ethanol (EtOH^R). EtOH^R cells were cross-resistant to 1% (vol/vol) methanol and 1% (vol/vol) 2-propanol but not 1% (vol/vol)n-propanol orn-butanol, whereas wild type (WT) cells were resistant only to methanol. Resistance in EtOH^R cells is probably a result of a very low level of alcohol dehydrogenase (ADH) activity which was only 9 to 10% of that in WT cells and was undetectable during much of the EtOH^R growth cycle. In the absence of ethanol, EtOH^R cells have a I₅₀ for the toxic ethanol analog allyl alcohol, which is nearly 3 times higher than that in WT cells. In the presence of ethanol, EtOH^R cells have an I₅₀ for allyl alcohol which is 12 times more than WT cells. This difference correlated well with the decrease in ADH activity found in EtOH^R cells grown on ethanol. When ethanol was removed from the suspension medium, ADH activity in EtOH^R cells gradually increased to WT levels. When re-exposed to ethanol after 200 cell generations, ADH activity quickly decreased and growth resumed after a 4- to 6-day lag period. Lipid analysis showed a 37% increase in total lipid in EtOH^R cells, mostly in polar lipids, di- and triglycerides. The fatty acid composition of these lipid classes was shifted toward the more polyunsaturated. These lipid changes were probably a reflection of the increased plastid number in the EtOH^R cells and may be a result of growth in ethanol rather than a reason for resistance. EtOH^R cells seem to be regulatory mutants able to quickly lower ADH activity in the presence of ethanol.     

Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas

A murine endothelial cell line was isolated from hemangiomas induced by expression of the polyoma early region gene in transgenic mice. After two cell sortings using acetylated low-density lipoprotein with a fluorescent label (Dil-Ac-LDL), a pure population of endothelial cells has been carried for more than 60 passages from the animal. The cells retain endothelial cell properties such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, and active uptake of Ac-LDL. Expression analysis shows that the cells express both the polyoma transgene and the von Willebrand factor, an endothelial cell marker. Subcutaneous injection of the cultured endothelial cells into nontransgenic histocompatible mice or nude mice led to hemangioma formation, and endothelial cells were re-isolated by cell sorting from these secondary hemangiomas. This cell line represents a renewable source of murine endothelial cells derived from transgenic mice that can be studied both in vitro and by reintroduction into a host.     

Isolation and characterization of NaCl resistant cell line of mulberry

The selected NaCl tolerant clones ofMorus alba L. cv. MR₂ grow better at higher concentration of NaCl than non-selected clones. With increasing NaCl concentration the Na⁺, Cl^? and proline content increased more and K⁺ and Ca²⁺ content decrease less in selected clones in comparison with non-selected ones.     

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.     

Establishment and characterization of an abrin-resistant cell line

An abrin-resistant cell line, CHOR 3-4, was isolated from CHOK1 cells which were resistant to a high concentration of abrin (160 ng/ml), and had a 1000-fold higher resistance to abrin that that of CHOK1 cells. CHOR 3-4 cells were about 25-fold more resistant than CHOK1 cells to the N-glycosidase activity of abrin, which was measured by hydrolyzing the N-glycosidic bond of adenine 4324 nucleotide from 3′ end of mammalian 28S rRNA. However, the isolated polysomes of CHOR 3-4 cells had the same sensitivity to abrin as those of CHOK1 cells. On measuring the binding of 125I-abrin to CHOR 3-4 cells, it was decreased to about 20% that of CHOK1 cells. This indicates that the mechanism of the resistance of CHOR 3-4 to abrin is due to the alteration of glycoproteins or glycolipids of cell membrane.     

Isolation and characterization of an anodic form of human liver alcohol dehydrogenase

A form of human liver alcohol dehydrogenase previously identified on starch gel electrophoresis as the anodic band (Li, T.-K. and Magnes, L.J. Biochem. Biophys. Res. Commun. 63, 202, 1975) has now been separated from the other molecular forms of the enzyme by affinity chromatography on 4-[3-(N-6-aminocaproyl)-aminopropyl]-pyrazole-Sepharose and purified to homogeneity on Agarose-hexane-AMP. Its physical properties are similar to those of other molecular forms already known, suggesting that they may be related. In contrast to other forms, the anodic species is inactive towards methanol, and its K_M for ethanol is as much as 100 times that of the other forms. This anodic form of alcohol dehydrogenase may contribute significantly to alcohol elimination in man, particularly at high alcohol concentrations when the other enzyme species are saturated.     

Isolation and characterization of a novel inducible mammalian galectin

A novel mammalian galectin cDNA (ovgal11) was isolated by representational difference analysis from sheep stomach (abomasal) tissue infected with the nematode parasite, Haemonchus contortus. The mRNA is greatly up-regulated in helminth larval infected gastrointestinal tissue subject to inflammation and eosinophil infiltration. Immunohistological analysis indicates that the protein is localized in the cytoplasm and nucleus of upper epithelial cells of the gastrointestinal tract. The protein is also detected in mucus samples collected from infected abomasum but not from uninfected tissue. The restricted and inducible expression of ovgal11 mRNA and limited secretion of the protein support the hypothesis that OVGAL11 may be involved in gastrointestinal immune/inflammatory responses and possibly protection against infection.     

Process development for an inducible rituximab-expressing Chinese hamster ovary cell line

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 ⁶ cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 ⁶ cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019     

Establishment and characterization of an immortalized epicardial cell line

Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40-LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.     

Isolation and molecular characterization of a mouse renal microvascular endothelial cell line

Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle alpha-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13-20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.     

Isolation and characterization of a neural progenitor cell line from tilapia brain

Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.     

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive (ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed.Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.     

Isolation and characterization of the highly immunogenic cell wall-associated protein of Mycobacterium leprae

In a recent study, we demonstrated that certain reactivities crucial to the immune response in leprosy are due to protein associated with the cell wall peptidoglycan "core" of Mycobacterium leprae. We now describe a primary method for the isolation of a highly immunogenic, large molecular-size, cell wall protein (CW-P) complex from M. leprae, freed of soluble proteins, bound mycolates, arabinogalactan, and much of the peptidoglycan. The complex is of apparent relative molecular size 2 x 10(6) to 20 x 10(6) Da, is distinguished by a high content of Ala, Gly, Leu, Asx, and Glx, and some peptidoglycan, and represents up to 7% of the bacterial mass. It is stable to a variety of dissociation and reductive processes and, in accord with its size, is not resolvable by polyacrylamide gel electrophoresis. The mAb to the CW-P complex also react with the heat shock 65-kDa protein of M. leprae. Conversely, antibodies that recognize internal epitopes within the polypeptide chain of the heat shock protein also react with CW-P; however, antibodies that recognize the N and C termini of the 65-kDa protein fail to react with CW-P, and some anti-CW-P mAb do not recognize any of the soluble proteins of M. leprae. Alternate methods to derive the large peptidoglycan-associated protein result in lower yield and less of the associated heat shock protein, implying that the 65-kDa protein may not be crucial to the immunogenicity of the complex. In an accompanying paper, we demonstrate that T cell clones raised to CW-P also selectively recognize soluble proteins, primarily of 7-kDa and 16-kDa size. Thus, the image of the CW-P complex of M. leprae is of a few immunoreactive polypeptides in avid association with a modicum of peptidoglycan to which the 65-kDa polypeptide may be variably attached, perhaps due to involvement in assembly of the complex.