scholarly journals Egg White Alginate as a Novel Scaffold Biomaterial for 3D Salivary Cell Culturing

Biomimetics ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 5
Author(s):  
Hieu M. Pham ◽  
Yuli Zhang ◽  
Jose G. Munguia-Lopez ◽  
Simon D. Tran
Keyword(s):  
Salivary Gland ◽  
Cell Viability ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Egg White ◽  
Simian Virus ◽  
Dead Cell ◽  
Salivary Cell ◽  
Novel Scaffold ◽  
Saliva Production

Saliva production by salivary glands play a crucial role in oral health. The loss of salivary gland function could lead to xerostomia, a condition also known as dry mouth. Significant reduction in saliva production could lead to further complications such as difficulty in speech, mastication, and increased susceptibility to dental caries and oral infections and diseases. While some palliative treatments are available for xerostomia, there are no curative treatments to date. This study explores the use of Egg White Alginate (EWA), as an alternative scaffold to Matrigel® for culturing 3D salivary gland cells. A protocol for an optimized EWA was established by comparing cell viability using 1%, 2%, and 3% alginate solution. The normal salivary simian virus 40-immortalized acinar cell (NS-SV-AC) and the submandibular gland-human-1 (SMG-hu-1) cell lines were also used to compare the spheroid formation and cell viability properties of both scaffold biomaterials; cell viability was observed over 10 days using a Live–Dead Cell Assay. Cell viability and spheroid size in 2% EWA was significantly greater than 1% and 3%. It is evident that EWA can support salivary cell survivability as well as form larger spheroids when compared to cells grown in Matrigel®. However, further investigations are necessary as it is unclear if cultured cells were proliferating or aggregating.

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells

1991 ◽  
Vol 11 (2) ◽  
pp. 1023-1029
Author(s):  
Y Li ◽  
D Li ◽  
K Osborn ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Housekeeping Gene ◽  
Simian Virus ◽  
Coding Region ◽  
Proliferating Cells ◽  
Early Promoter

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

1988 ◽  
Vol 8 (1) ◽  
pp. 62-70
Author(s):  
J B Jaynes ◽  
J E Johnson ◽  
J N Buskin ◽  
C L Gartside ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Major Influence ◽  
Transcriptional Enhancer ◽  
Muscle Creatine Kinase ◽  
Kinase Gene ◽  
Muscle Creatine

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. We have examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

scholarly journals Fodrin: axonally transported polypeptides associated with the internal periphery of many cells.

1981 ◽  
Vol 90 (3) ◽  
pp. 631-642 ◽  
Author(s):  
J Levine ◽  
M Willard
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cortical Actin ◽  
Cultured Fibroblasts ◽  
Tissue Sections ◽  
Parallel Arrays ◽  
Guinea Pig Brain

Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d--slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins. In this communication, we report the intracellular distribution of fodrin. Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence. Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e.g., skeletal muscle, uterus, intestinal epithelium). Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining). In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro. We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities: (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm; and (c) that fodrin could serve to link other proteins and organelles to a submembrane force-generating system.

scholarly journals Quantitative analysis of intercellular adhesive specificity in freshly explanted and cultured cells.

1981 ◽  
Vol 90 (1) ◽  
pp. 55-62 ◽  
Author(s):  
F Sieber ◽  
S Roseman
Keyword(s):  
Quantitative Analysis ◽  
Cho Cells ◽  
Binomial Distribution ◽  
Cultured Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Fluorescent Label ◽  
3T3 Cells

A new method is presented for the quantitative analysis of intercellular adhesive specificity. In this assay, two cell types are mixed, one unlabeled and the other labeled with the fluorescent dye, fluorescamine [4-phenylspiro(feran-2[3H],1'-phthalan)-3,3'-dione]. The resulting aggregates are analyzed by fluorescence microscopy to determine the number of labeled and unlabeled cells per aggregate. Random (nonspecific) aggregation was characterized by a binomial distribution, and adhesive specificity was accordingly quantified by the deviation (as determined by a chi-square test) from the calculated binomial distribution. The labeling procedure was simple and rapid, and experiments with 18 different cell types showed that it did not affect cell viability, morphology, rate and extent of adhesion, plating efficiency, and the capability of myogenic cells to undergo terminal differentiation. Most important, assays with morphologically identifiable cell pairs indicated that the fluorescent label neither induced apparent nor destroyed existing adhesive specificity. The most pronounced adhesive specificities were observed with freshly explanted cells from adult tissues and also with mixtures of simian virus 40-transformed and nontransformed BALB/c 3T3 cells. A glucosamine-6-phosphate N-acetylase-deficient mutant 3T3 line (AD6), however, aggregated randomly with parental 3T3 cells. Lectin-resistant mutant Chinese hamster ovary (CHO) cells displayed marginal adhesive specificity when mixed with normal CHO cells.

scholarly journals The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.

1991 ◽  
Vol 11 (2) ◽  
pp. 1023-1029 ◽  
Author(s):  
Y Li ◽  
D Li ◽  
K Osborn ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Housekeeping Gene ◽  
Simian Virus ◽  
Coding Region ◽  
Proliferating Cells ◽  
Early Promoter

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

scholarly journals The transcriptome of wild-type and immortalized corneal epithelial cells

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Kai Furuya ◽  
Tao Wu ◽  
Ai Orimoto ◽  
Eriko Sugano ◽  
Hiroshi Tomita ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Expression Pattern ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial

AbstractCellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.

Failure to detect papovavirus-associated T antigens in human brain tumor cells by anticomplement immunofluorescence

1980 ◽  
Vol 52 (3) ◽  
pp. 367-370 ◽  
Author(s):  
Hideyuki Kosaka ◽  
Yoshinori Sano ◽  
Yasuhiko Matsukado ◽  
Takeshi Sairenji ◽  
Yorio Hinuma
Keyword(s):  
Brain Tumors ◽  
Human Brain ◽  
Cultured Cells ◽  
Jc Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Bk Virus ◽  
Tumor Promoter ◽  
Human Brain Tumor

✓ To probe the possible presence of papovavirus-related T antigen(s) in human brain tumors, the imprinted or cultured cells at various passage levels were examined by anticomplement immunofluorescence using antisera to T antigen of each BK virus, JC virus, and simian virus 40. No T antigen was demonstrated in any tests with cells derived from 69 patients with various brain tumors. Twenty-two tumor cell strains cultured in the presence of a tumor promoter, phorbol ester, also failed to show the T antigen.

scholarly journals Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

1985 ◽  
Vol 5 (6) ◽  
pp. 1247-1259 ◽  
Author(s):  
J R Stringer
Keyword(s):  
Homologous Recombination ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Major Pathway ◽  
Viral Genomes ◽  
Nonhomologous Recombination ◽  
Adenylic Acid ◽  
Sv40 Dna

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.

scholarly journals Intestinal Hyperplasia Induced by Simian Virus 40 Large Tumor Antigen Requires E2F2

2007 ◽  
Vol 81 (23) ◽  
pp. 13191-13199 ◽  
Author(s):  
M. Teresa Sáenz-Robles ◽  
Jennifer A. Markovics ◽  
Jean-Leon Chong ◽  
Rene Opavsky ◽  
Robert H. Whitehead ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
Large Tumor ◽  
Intestinal Hyperplasia

ABSTRACT The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.

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