Saline Solution Effects on Propidium Iodide Uptake in Nanoinjected HeLa Cells

Being able to deliver molecular loads to the intracellular space of mammalian cells is a key initial step of genetic engineering. In the following work, experimentation with nanoinjection, a non-viral molecular load delivery technique, was examined in regards to transmembrane delivery of propidium iodide (PI), a dye that cannot penetrate the cell membrane and fluoresces when bound to genetic material. Investigation includes two environmental factors: peak pulse amplitude (1.5 to 3, 5, 7, or 9 V) and saline type (HBSS, PBS with potassium, and PBS without potassium). Results indicate that PBS with potassium has significantly higher PI uptake efficiency than the other two saline solutions for pulsed voltages of 3V, 5V, and 7V (with the peak value being 3.352 times greater than the positive control). Also, cell viability analysis indicates that there is a measureable reduction in cell viability for voltage protocol samples in comparison to non-voltage protocol samples. Cell viabilities range from 74.5% to 89.4% for voltage protocol samples. Findings suggest that a possible combination of physical/electrical variables work in concert with biological mechanisms to contribute to overall cell survival and PI uptake efficiency in nanoinjection.

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Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

Chemical Communications ◽

10.1039/c4cc08862j ◽

2015 ◽

Vol 51 (37) ◽

pp. 7887-7890 ◽

Cited By ~ 17

Author(s):

Hideto Maruyama ◽

Kazuhiro Furukawa ◽

Hiroyuki Kamiya ◽

Noriaki Minakawa ◽

Akira Matsuda

Keyword(s):

Nucleic Acids ◽

Mammalian Cells ◽

Genetic Material ◽

Rna Polymerases ◽

Chemically Modified ◽

Biological Studies ◽

Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

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Effect of intracellular uptake of nanoparticle-encapsulated trehalose on the hemocompatibility of allogeneic valves in the VS83 vitrification protocol

Nanobiomedicine ◽

10.1177/1849543520983173 ◽

2020 ◽

Vol 7 ◽

pp. 184954352098317

Author(s):

Balamurugan Vasudevan ◽

Qing Chang ◽

Bin Wang ◽

Siyang Huang ◽

Yulong Sui ◽

...

Keyword(s):

Cell Viability ◽

Heart Valves ◽

Corn Starch ◽

Colloidal Suspensions ◽

Initial Step ◽

Control Group ◽

Hydroxyapatite Nanoparticles ◽

Reticular Fibers ◽

Terminal Complement ◽

Ammonia Method

Trehalose is a disaccharide molecule consisting of two molecules of glucose. Industrially, trehalose is derived from corn starch and utilized as a drug. This study aims to examine whether the integration of nanoparticle-encapsulated trehalose to the Ice-Free Cryopreservation (IFC) method for preserving heart valves has better cell viability, benefits to protect the extracellular matrix (ECM), and reduce immune response after storage. For the experiment to be carried out, we obtained materials, and the procedures were carried out in the following manner. The initial step was the preparation of hydroxyapatite nanoparticles, followed by precipitation to acquire Apatite colloidal suspensions. Animals were obtained, and their tissue isolation and grouping were done ethically. All samples were then divided into four groups, Control group, Conventional Frozen Cryopreservation (CFC) group, IFC group, and IFC + T (IFC with the addition of 0.2 M nanoparticle-encapsulated Trehalose) group. Histological analysis was carried out via H&E staining, ECM components were stained with Modified Weigert staining, and the Gomori Ammonia method was used to stain reticular fibers. Alamar Blue assay was utilized to assess cell viability. Hemocompatibility was evaluated, and samples were processed for immunohistochemistry (TNFα and IL-10). Hemocompatibility was quantified using Terminal Complement Complex (TCC) and Neutrophil elastase (NE) as an indicator. The results of the H&E staining revealed less formation of extracellular ice crystals and intracellular vacuoles in the IFC + T group compared with all other groups. The CFC group’s cell viability showed better viability than the IFC group, but the highest viability was exhibited in the IFC + T group (70.96 ± 2.53, P < 0.0001, n = 6). In immunohistochemistry, TNFα levels were lowest in both IFC and IFC + T group, and IL-10 expression had significantly reduced in IFC and IFC + T group. The results suggested that the nanoparticle encapsulated trehalose did not show significant hemocompatibility issues on the cryopreserved heart valves.

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Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

The Journal of Cell Biology ◽

10.1083/jcb.115.1.31 ◽

1991 ◽

Vol 115 (1) ◽

pp. 31-43 ◽

Cited By ~ 235

Author(s):

H Plutner ◽

A D Cox ◽

S Pind ◽

R Khosravi-Far ◽

J R Bourne ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Essential Role ◽

Secretory Pathway ◽

Binding Protein ◽

Vesicular Transport ◽

Initial Step ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

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Correction of Genetic Defects in Mammalian Cells by the Input of Small Amounts of Foreign Genetic Material

Journal of Cell Science ◽

10.1242/jcs.13.3.841 ◽

1973 ◽

Vol 13 (3) ◽

pp. 841-861

Author(s):

YVONNE L. BOYD ◽

H. HARRIS

Keyword(s):

Thymidine Kinase ◽

Mammalian Cells ◽

Hybrid Cell ◽

Genetic Material ◽

Chinese Hamster ◽

Heat Sensitivity ◽

Chinese Hamster Cells ◽

Inosinic Acid ◽

Mouse Cells

Chinese hamster cells lacking inosinic acid pyrophosphorylase and mouse cells lacking thymidine kinase were fused with chick erythrocytes. The resultant heterokaryons were cultivated in a selective medium in which possession of these enzymes was essential for cell survival and growth. Clones of cells able to grow in this medium were isolated and studied. A detailed karyological analysis of these clones failed to reveal any chick chromosomes; nor could any chick-specific antigens be detected on the surface of the cells. Nonetheless, clones arising from the fusion of chick erythrocytes with Chinese hamster cells were shown to possess an inosinic acid pyrophosphorylase which had the electrophoretic characteristics of chick inosinic acid pyrophosphorylase. However, the clones arising from the fusion of the chick erythrocytes with the mouse cells had a thymidine kinase with the electrophoretic mobility and heat sensitivity of murine, not chick, thymidine kinase. Both types of hybrid cell have now been cultivated in vitro for 18 months without the loss of thymidine kinase or inosinic acid pyrophosphorylase activity.

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Effect of ZnO nanoparticle on cell viability, zinc uptake efficiency, and zinc transporters gene expression: a comparison with ZnO and ZnSO4

Czech Journal of Animal Science ◽

10.17221/15/2016-cjas ◽

2017 ◽

Vol 62 (No. 1) ◽

pp. 32-41 ◽

Cited By ~ 2

Author(s):

X. Zhang ◽

Z. Wang ◽

L. Mao ◽

X. Dong ◽

Q. Peng ◽

...

Keyword(s):

Gene Expression ◽

Zinc Oxide ◽

Cell Viability ◽

Feed Additives ◽

Zinc Uptake ◽

Zinc Transporters ◽

Zno Nanoparticle ◽

Nano Zno ◽

Uptake Efficiency ◽

Zinc Sources

Zinc plays an important role in functional and structural integrity of cells. The aim of the current study was to compare cell viability, zinc uptake efficiency, and gene expression of metallothionein (MT), divalent metal transporter (DMT-1), and other important zinc transporters (ZnTs) under experimental treatment of TPEN (N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine) (2 µM), and three zinc sources (zinc oxide nanoparticle (nano-ZnO), bulk zinc oxide (ZnO), and zinc sulfate (ZnSO4)) at different levels (25, 50, and 100 µM) in rat intestinal epithelial cell line IEC-6. Cells were classified into TPEN group and TPEN + zinc sources groups. In the present study, significantly decreased cell viability was observed in TPEN group, while supplementations with nano-ZnO at all levels and ZnO (50 and 100 µM) significantly increased the cell viability. ZnSO4 at a high concentration (100 µM) inhibited cell viability. Furthermore, cells of nano-ZnO group showed the highest viability at a 25 µM concentration. The uptake efficiency of nano-ZnO is higher than that of ZnSO4 and ZnO. Additionally, a significant down-regulation for ZnT-1, ZnT-4, MT, DMT-1 mRNA with TPEN treatment was detected. Compared with the unchanged ZnT-4, all zinc treatments up-regulated the gene expressions of ZnT-1, ZnT-5, ZnT-7, MT, and DMT-1. Our results indicate that nano-ZnO is more effective than ZnO and ZnSO4 in enhancing cell viability, and its lower cytotoxicity, higher uptake efficiency, and comparative transportation at low concentration also favour its potential use as a new zinc source in feed additives.

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Androgen negative feedback during the early rise in LH secretion in bull calves

Journal of Endocrinology ◽

10.1677/joe.0.1450243 ◽

1995 ◽

Vol 145 (2) ◽

pp. 243-249 ◽

Cited By ~ 29

Author(s):

N C Rawlings ◽

A C O Evans

Keyword(s):

Sexual Maturity ◽

Initial Step ◽

Pulse Amplitude ◽

Pulse Frequency ◽

Serum Samples ◽

Bull Calf ◽

Bull Calves ◽

Early Increase ◽

Early Rise ◽

Lh Secretion

Abstract A transient elevation in mean circulating concentrations of LH and FSH occurs in the young bull calf prior to 24 weeks of age. The functional significance of this is not clear. To see if changes in the ability of androgens to suppress gonadotrophin secretion were involved in the start of this early rise in LH secretion or the cessation of the early rise in LH and FSH secretion, bull calves were treated with flutamide (androgen receptor blocker; n=5; 9 mg flutamide/kg body weight in propylene glycol (i.m./s.c.) in three equal portions at 12-h intervals) at 8, 16 and 24 weeks of age and bled every 15 min for 12 h beginning after the third flutamide treatment; control bulls received vehicle at these times. Control bulls (n=5) were bled every 15 min for 12 h at 4, 8, 12, 16 and 24 weeks of age, and all bulls were bled weekly. Serum samples were assayed for concentrations of LH, FSH and testosterone. Based on weekly and intensive bleedings for control and flutamide-treated bulls, an early rise in LH (8–18 weeks of age) and FSH (4–24 weeks of age) secretion was seen in all bull calves (P<0·05). At 8 weeks of age flutamide treatment resulted in increased mean serum LH concentrations (P<0·05); at 16 weeks of age it resulted in increased basal and mean LH concentrations and increased LH pulse frequency (P<0·05); and at 24 weeks of age in increased mean LH concentrations, LH pulse frequency and amplitude (P<0·05) in comparison with control bulls. Flutamide treatment resulted in decreased FSH pulse amplitude at 8 weeks of age and increased mean serum concentrations of FSH and FSH pulse frequency at 24 weeks of age (P<0·05). In flutamide-treated bull calves testicular growth was greater and sexual maturity was reached earlier than in control bull calves (P<0·05). We conclude that a reduced suppression of LH secretion by androgens does not appear to be a major contributing factor to the onset of the early increase in LH secretion, but increased suppression may be involved in the termination of the early rise of both LH and FSH secretion in the bull calf. The early increase in LH secretion may be a critical initial step in postnatal reproductive development, since flutamide treatment increased early LH secretion and resulted in earlier attainment of sexual maturity. Journal of Endocrinology (1995) 145, 243–249

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Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724303209049 ◽

2003 ◽

Vol 2003 (2) ◽

pp. 79-91 ◽

Cited By ~ 20

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.

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Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity

Biochemical Journal ◽

10.1042/bcj20180155 ◽

2018 ◽

Vol 475 (10) ◽

pp. 1773-1788 ◽

Cited By ~ 4

Author(s):

Ditlev Birch ◽

Malene V. Christensen ◽

Dan Staerk ◽

Henrik Franzyk ◽

Hanne Mørck Nielsen

Keyword(s):

Amino Acids ◽

Cell Viability ◽

Mammalian Cells ◽

Membrane Integrity ◽

Cell Penetrating Peptides ◽

Proliferating Cells ◽

Epithelial Integrity ◽

Cell Penetrating ◽

Culture Models ◽

Well Differentiated

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18–20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

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Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation

Biomolecules ◽

10.3390/biom9050186 ◽

2019 ◽

Vol 9 (5) ◽

pp. 186 ◽

Cited By ~ 3

Author(s):

Sofia Piña Olmos ◽

Roberto Díaz Torres ◽

Eman Elbakrawy ◽

Louise Hughes ◽

Joseph Mckenna ◽

...

Keyword(s):

Breast Cancer ◽

Cell Viability ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Spindle Assembly Checkpoint ◽

Genome Instability ◽

Chitosan Nanoparticles ◽

Genetic Material ◽

X Ray ◽

Before And After

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

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