Localization of Z-RNA in Normal Lens Epithelium: Middle Fibers

Our goal was to determine the cellular localization of left-handed Z-RNA, within preeguatorial zone (PZ) epithelium of the normal adult dog ocular lens (1.5 yr) (Fig. 1), employing anti-Z-RNA IgG polyclonal antibodies. B-DNA has the ability to adopt the Z-DNA configuration in vitro(1). A-RNA can be transformed into Z-RNA under certain conditions (2). Z-RNA has been localized in cultured cells (3). Strong evidence supports the presence of Z-DNA in vivo (1). Elimination of DNA binding proteins by certain fixatives can initiate DNA supercoiling which stabilizes Z-DNA sequences (1). Z-DNA may play a role in regulating in vivo transcriptional enhancement (1).Anti-Z-RNA antibody probes were produced in 3 rabbits immunized with injections of Z-RNA: Br-poly[ribosomal(G-C)]. Concerning light microscopy [immunohistochemistry (ABC method)], lens tissues were fixed in Carnoy's, embedded in paraffin and sectioned (3 μm) (Fig. 2). Image analysis was performed using a Leitz DM-RB microscope and Leica Quantiment 500 + image analyzer.

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Demonstration of Z-RNA in the Dog Eye Lens Epithelium (Germinative Zone)

Microscopy and Microanalysis ◽

10.1017/s1431927600025666 ◽

1998 ◽

Vol 4 (S2) ◽

pp. 1108-1109

Author(s):

C.E. Gagna ◽

J.H. Chen ◽

H.R. Kuo ◽

W.C. Lambert

Keyword(s):

Cellular Localization ◽

Cultured Cells ◽

Polyclonal Antibodies ◽

Phosphate Buffer Solution ◽

Buffer Solution ◽

Solution Ph ◽

Immunogold Staining ◽

Z Dna

The purpose of this scientific investigation was to determine the presence and specific cellular localization of left-handed Z-RNA, within germinative zone (GZ) epithelium of the lens (Fig. 1), using anti-Z-RNA IgG polyclonal antibodies. Right-handed B-DNA has the ability to adopt the Z-DNA conformation in vitro (Sinden, 1994). Right-handed A-RNA can be transformed into Z-RNA under specific conditions (Hall et al., 1984), and Z-RNA has been identified in cultured cells (Zarling et al., 1990). Strong evidence supports the idea of Z-DNA in vivo (Sinden, 1994). Removal of proteins by fixatives can induce supercoiling which stabilizes Z-DNA (Sinden, 1994).Anti-Z-RNA antibodies were produced in rabbits immunized with injections of Z-RNA: brominated-poly[ribosomal(G-C)]. For light microscopy, immunohistochemical studies (ABC method), normal dog lens tissues (1 yr old) were fixed in Carnoy's, embedded in paraffin and sectioned 2 μm thick. For electron microscopy (immunogold staining), pieces of epithelium from the GZ of normal dog lens (1 yr old) were fixed with 5% glutaraldehyde in 0.05 M phosphate buffer solution, pH 7.3.

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Differential Stabilization of Left-Handed Z-DNA and Z-RNA In Vitro and In Vivo

Enzyme Dynamics and Regulation ◽

10.1007/978-1-4612-3744-0_23 ◽

1988 ◽

pp. 190-199 ◽

Cited By ~ 1

Author(s):

Thomas M. Jovin ◽

Donna J. Arndt-Jovin

Keyword(s):

Left Handed ◽

Z Dna

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Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5679 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5679-5687 ◽

Cited By ~ 148

Author(s):

C P Chang ◽

Y Jacobs ◽

T Nakamura ◽

N A Jenkins ◽

N G Copeland ◽

...

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Cultured Cells ◽

Binding Activity ◽

Wild Type ◽

Hox Proteins ◽

Amino Terminal ◽

Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

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The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.785-793.1990 ◽

1990 ◽

Vol 10 (2) ◽

pp. 785-793

Author(s):

W P Wahls ◽

L J Wallace ◽

P D Moore

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Dna Sequences ◽

Human Cells ◽

Base Pairs ◽

Cells In Culture ◽

Left Handed ◽

Dna Motif ◽

Z Dna

Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.

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The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture.

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.785 ◽

1990 ◽

Vol 10 (2) ◽

pp. 785-793 ◽

Cited By ~ 82

Author(s):

W P Wahls ◽

L J Wallace ◽

P D Moore

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Dna Sequences ◽

Human Cells ◽

Base Pairs ◽

Cells In Culture ◽

Left Handed ◽

Dna Motif ◽

Z Dna

Tracts of the alternating dinucleotide polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout the human genome, are capable of readily forming left-handed Z-DNA in vitro. We have analyzed the effects of the Z-DNA motif d(TG)30 upon homologous recombination between two nonreplicating plasmid substrates cotransfected into human cells in culture. In this study, the sequence d(TG)30 is shown to stimulate homologous recombination up to 20-fold. Enhancement is specific to the Z-DNA motif; a control DNA fragment of similar size does not alter the recombination frequency. The stimulation of recombination is observed at a distance (237 to 1,269 base pairs away from the Z-DNA motif) and involves both gene conversion and reciprocal exchange events. Maximum stimulation is observed when the sequence is present in both substrates, but it is capable of stimulating when present in only one substrate. Analysis of recombination products indicates that the Z-DNA motif increases the frequency and alters the distribution of multiple, unselected recombination events. Specifically designed crosses indicate that the substrate containing the Z-DNA motif preferentially acts as the recipient of genetic information during gene conversion events. Models describing how left-handed Z-DNA sequences might promote the initiation of homologous recombination are presented.

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Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053711 ◽

1982 ◽

Vol 40 ◽

pp. 210-211

Author(s):

M.J. Murphy ◽

R.R. Price ◽

J.C. Sloman

Keyword(s):

Cultured Cells ◽

Human Tumor ◽

Cell Type ◽

Tumor Mass ◽

Initial Cell ◽

Cloning Assay ◽

Human Tumor Cloning ◽

The Relationship

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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Preparation of cultured astrocytes for x-ray microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156924 ◽

1989 ◽

Vol 47 ◽

pp. 988-989

Author(s):

N.K.R. Smith ◽

K.E. Hunter ◽

P. Mobley ◽

L.P. Felpel

Keyword(s):

Cultured Cells ◽

Elemental Content ◽

Elemental Distribution ◽

Sprague Dawley ◽

X Ray ◽

Cultured Astrocytes ◽

Freeze Dried ◽

Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

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TAMI-06. PRECLINICAL MODELS REVEAL BRAIN-MICROENVIRONMENT SPECIFIC METABOLIC DEPENDENCIES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.895 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii214-ii214

Author(s):

Jenna Minami ◽

Nicholas Bayley ◽

Christopher Tse ◽

Henan Zhu ◽

Danielle Morrow ◽

...

Keyword(s):

Tumor Microenvironment ◽

Cultured Cells ◽

Tumor Biology ◽

Metabolic Reprogramming ◽

Neoplastic Progression ◽

Extracellular Milieu ◽

Metabolic Defects ◽

Metabolic Heterogeneity

Abstract Metabolic reprogramming is a hallmark of cancer, and malignant cells must acquire metabolic adaptations to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Conventional cell lines do not recapitulate the metabolic heterogeneity of glioblastoma (GBM), while primary cultured cells do not account for the influences of the microenvironment and the blood brain barrier on tumor biology. Additionally, these systems are under strong selective pressure divergent from that in vivo, leading to reduced heterogeneity between cultured tumor cells. Here, we describe a biobank of direct-from-patient derived orthotopic xenografts (GliomaPDOX) and gliomaspheres that reveal a subset of gliomas that, while able to form in vivo, cannot survive in vitro. RNA sequencing of tumors that can form both in vivo and in vitro (termed “TME-Indifferent”) compared to that of tumors that can only form in vivo (termed “TME-Dependent”) revealed transcriptional changes associated with altered nutrient availability, emphasizing the unique metabolic programs impacted by the tumor microenvironment. Furthermore, TME-dependent tumors lack metabolic signatures associated with nutrient biosynthesis, thus indicating a potential dependency of these tumors on scavenging specific nutrients from the extracellular milieu. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity, indicating a potential brain-microenvironment specific metabolic dependency that can be targeted for therapy.

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Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

Scientific Reports ◽

10.1038/s41598-021-93607-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shojiro Katoh ◽

Atsuki Fujimaru ◽

Masaru Iwasaki ◽

Hiroshi Yoshioka ◽

Rajappa Senthilkumar ◽

...

Keyword(s):

Regenerative Medicine ◽

Replicative Senescence ◽

Cultured Cells ◽

Three Dimensional ◽

Human Chondrocytes ◽

Cartilage Tissue ◽

Optimal Method ◽

Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

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