scholarly journals Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 145-153 ◽  
Author(s):  
Sojung Kwon ◽  
Areum Kwak ◽  
Hyejin Shin ◽  
Soyoung Choi ◽  
Soohyun Kim ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Hela Cells ◽  
Protein Delivery ◽  
Cancer Cell Line ◽  
Preimplantation Embryos ◽  
Cervical Cancer Cell Line ◽  
Cell Stage ◽  
Cell Permeable Peptide ◽  
Morula Stage ◽  
Punctate Pattern

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12–EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12–EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12–EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP–LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP–LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.

Nuclear Channels in Hela Cells: Nucleocytoplasmic Transport or Reassembly of the Nuclear Envelope?

2001 ◽  
Vol 7 (S2) ◽  
pp. 640-641
Author(s):  
J.R. Palisano ◽  
E.K. Traister
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Fluorescent Microscopy ◽  
Calf Serum ◽  
Cancer Cell Line ◽  
Nucleocytoplasmic Transport ◽  
Culture Collection ◽  
Cervical Cancer Cell Line ◽  
Electron Microscopic ◽  
And Function

Although nuclear channels (NC) have been described in a variety of cells, their function has not been fully investigated. This investigation used immunofluorescent techniques to examine the formation and function of NC in HeLa cells, a human cervical cancer cell line. NC are defined as invaginations of the nuclear envelope (NE) that traverse the nucleoplasm, thus forming a ring-like nucleus. The resulting tunnel is devoid of nucleic acid and may contain cytoplasmic elements. to date, all the reports of the presence of NC are from electron microscopic investigations, which clearly demonstrate the occurrence of NC in the presence of what appear to be interphase nuclei. This investigation was initiated because the NC that we observed in HeLa cells could be seen with fluorescent microscopy and only appeared during telophase and early interphase.HeLa cells, obtained from American Type Culture Collection, were cultured on glass coverslips in minimum essential medium (MEM) containing 5% calf serum (CS) and a mixture of 5% CO2 in air at 37°C.

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 147-158 ◽  
Author(s):  
Shangdan Xu ◽  
Jibak Lee ◽  
Masashi Miyake
Keyword(s):  
Western Blotting ◽  
Contact Region ◽  
Relative Concentration ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Homogeneous Distribution ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Morula Stage

SummaryExpression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α− was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α− throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

scholarly journals Overexpression of the nucleoporin Nup88 stimulates migration and invasion of HeLa cells

2021 ◽  
Author(s):  
Masaki Makise ◽  
Ryota Uchimura ◽  
Kumiko Higashi ◽  
Yasumi Mashiki ◽  
Rikako Shiraishi ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cell ◽  
Hela Cells ◽  
Malignant Tumors ◽  
Cancer Cell Line ◽  
Migration And Invasion ◽  
Nuclear Pore ◽  
Cervical Cancer Cell Line ◽  
Invasive Ability ◽  
Mesenchymal Transition

AbstractElevated expression of the nucleoporin Nup88, a constituent of the nuclear pore complex, is seen in various types of malignant tumors, but whether this overexpression contributes to the malignant phenotype has yet to be determined. Here, we investigated the effect of the overexpression of Nup88 on the migration and invasion of cervical cancer HeLa cells. The overexpression of Nup88 promoted a slight but significant increase in both migration and invasion, whereas knockdown of Nup88 by RNA interference suppressed these phenotypes. The observed phenotypes in Nup88-overexpressing HeLa cells were not due to the progression of the epithelial-to-mesenchymal transition or activation of NF-κB, which are known to be important for cell migration and invasion. Instead, we identified an upregulation of matrix metalloproteinase-12 (MMP-12) at both the gene and protein levels in Nup88-overexpressing HeLa cells. Upregulation of MMP-12 protein by the overexpression of Nup88 was also observed in one other cervical cancer cell line and two prostate cancer cell lines but not 293 cells. Treatment with a selective inhibitor against MMP-12 enzymatic activity significantly suppressed the invasive ability of HeLa cells induced by Nup88 overexpression. Taken together, our results suggest that overexpression of Nup88 can stimulate malignant phenotypes including invasive ability, which is promoted by MMP-12 expression.

182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 190
Author(s):  
T.-Y. Fu ◽  
P.-C. Tang
Keyword(s):  
Expression Pattern ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Cell Stage ◽  
Morula Stage

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.

scholarly journals Bibenzyl Derivatives From Leaves of Dendrobium officinale

2020 ◽  
Vol 15 (2) ◽  
pp. 1934578X2090867
Author(s):  
Gang Ren ◽  
Wen-Zan Deng ◽  
Yan-Fei Xie ◽  
Chun-Hua Wu ◽  
Wen-Yan Li ◽  
...  
Keyword(s):  
Hela Cells ◽  
Inhibitory Concentration ◽  
Cancer Cell Line ◽  
Dendrobium Officinale ◽  
Cervical Cancer Cell Line ◽  
Cell Line Hela ◽  
Human Cervical Cancer Cell ◽  
Related Compounds ◽  
Line Hela ◽  
Human Cervical Cancer

Nine compounds were isolated from leaves of Dendrobium officinale, including 1 new bibenzyl derivative, denofficin (1), and 8 known structurally related compounds, dendrocandin B (2), dendrocandin U (3), 3,4-dihydroxy-5,4′-dimethoxy bibenzyl (4), moscatilin (5), 4,4′-dihydroxy-3,5-dimethoxy bibenzyl (6), ( S)-3,4,α-trihydroxy-5,4′-dimethoxy bibenzyl (7), gigantol (8), densiflorol A (9). The structures of these compounds were identified by spectroscopic methods. All isolated compounds were screened for their cytotoxicity against human cervical cancer cell line HeLa cells. Of them, compounds 1-3, 5, 6, and 8 were found to have the capabilities of proliferation inhibition against HeLa cells with half-maximal inhibitory concentration values ranging from 8.0 to 92.4 μM.

scholarly journals Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin

Reproduction ◽  
2003 ◽  
pp. 111-118 ◽  
Author(s):  
BD Higgins ◽  
MT Kane
Keyword(s):  
Gene Activation ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Sodium Dependent ◽  
Morula Stage ◽  
Zygotic Gene ◽  
Zygotic Gene Activation ◽  
The One ◽  
Inositol Uptake

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

scholarly journals The Anti-Cancer Property of Mumie as Natural Product on Human Cervical Cancer Cell Line (HeLa)

2020 ◽  
Vol 9 (1) ◽  
pp. 196-202
Keyword(s):  
Cervical Cancer ◽  
Hela Cells ◽  
Chromatin Condensation ◽  
Cancer Cell Line ◽  
Antioxidant Property ◽  
Cervical Cancer Cell Line ◽  
Cell Line Hela ◽  
Anti Cancer ◽  
Human Cervical Cancer Cell ◽  
Treated Culture

Mumie is a natural component found in some mountains, such as the Himalayas, as well as in some mountainous of Iran. It contains of humic and phenolic compounds that have antioxidant and anti-cancer properties. Therefore, in this study, anti-cancer and antioxidant properties of mumie were examined on Human Cervical Cancer Cell Line (HeLa). HeLa cells and normal fibroblasts (NIH) were cultured in DMEM/F12 with mumie at concentrations of 0, 100, 200, 300, 400, 500 and 1000 µg/ml for 24 and 48 h. The bioviability of these cells were evaluated using MTT assay. Chromatin condensation and apoptosis of these cells were examined using acridine orange and aniline blue staining respectively. Antioxidant property of mumie on NIH cells was evaluated by 10 mM H2O2 and neutral red test. MTT assay revealed bioviability of HeLa cells decreased but chromatin condensation increased in concentration of 100 μg/ml mumie treated culture. Apoptosis of the HeLa cells were observed in 100 μg/ml mumie treated culture. Mumie did not affect the bioviability, chromatin condensation and apoptosis of NIH cells but 500 and 1000 μg/ml concentrations were toxic and induced cell death. The cell cultures in different concentrations of mumie after 24and 48 h showed the similar results. NIH cells bioviability increased in 500 and 1000 μg/ml concentrations of co-culture of H2O2 and mumie that confirmed the antioxidant property. It concluded that even low concentrations of mumie could destroy HeLa cells without any side effect on normal cells. Therefore, it can be used for cervical cancer treatment but further research is needed.

scholarly journals CYTOTOXICITY AND APOPTOSIS INDUCTION BY KAFFIR LIME LEAVES EXTRACT (Citrus hystrix DC.) IN HeLa CELLS CULTURE (HUMAN CERVICAL CANCER CELL LINE)

2015 ◽  
Vol 2 (1) ◽  
pp. 631 ◽  
Author(s):  
Nastiti Wijayanti ◽  
W.A.S. Tunjung ◽  
Y. Setyawati
Keyword(s):  
Cervical Cancer ◽  
Ethyl Acetate ◽  
Cell Line ◽  
Cancer Cell ◽  
Hela Cells ◽  
Ethyl Acetate Extract ◽  
Cancer Cell Line ◽  
Cervical Cancer Cell ◽  
Cervical Cancer Cell Line ◽  
Cell Line Hela

<p>Kaffir lime (Citrus hystrix) is a native plant of Indonesia. Nowadays the use of plants is limited as ingredients in Indonesia cuisine and aromatherapy oils for industry. Leaves of C. hystrix contain of polyphenols and essential oils thus they were possibly cytotoxic to cancer cell line. The objective of this research was to determine the cytotoxicity effect and apoptosis induced by leaves extract C. hystrix to cervical cancer cell line (HeLa cells). Methods used in this study included sampling of kaffir lime leaves, extractionusing three different solvents (ethanol, ethyl acetate, and hexane), detection of metabolite secondary compound (alkaloids, flavonoids, terpenoids, saponins, and tannins) using thin layer chromatography (TLC), cytotoxicity assay via MTT assay, and apoptosis test with double staining method (ethidium bromide-acrydine orange). Result showed kaffir lime crude extract dose dependently inhibitHeLa cells proliferation. lC50of ethanolicand ethyl acetate extract was 82,034 µg/mL and 57,845 µg/mL, respectively means cytotoxic to HeLa cells. On the other hand lC50 of hexane extract was 203,992 µg/mL which was not cytotoxic. Furthermore ethanolic and ethyl acetate extract were able to induce apoptosis of HeLa cells by increasing the number of apoptotic cells. In conclusion, kaffir lime leaves extract had cytotoxic effect and induced apoptosis. Moreover ethyl acetate extractof kaffir lime was the most potential to induce apoptosis in HeLa cells. </p><p><strong>Keywords</strong>: kaffir lime leaves (Citrus hystrix), HeLa, MTT, cytotoxicity, apoptosis.</p>

294. Insulin receptor internalization in mouse preimplantation embryos

2005 ◽  
Vol 17 (9) ◽  
pp. 124
Author(s):  
F.-C. Hung ◽  
M. Pantaleon ◽  
P. L. Kaye
Keyword(s):  
Insulin Receptor ◽  
Receptor Internalization ◽  
Preimplantation Embryos ◽  
Regulation Of Transcription ◽  
Morphological Development ◽  
Cell Stage ◽  
Cytoplasmic Staining ◽  
Morula Stage ◽  
Exon 11

The insulin receptor (IR) mediates the actions of insulin and insulin-like growth factors (IGF-I and II). Two IR isoforms result from alternate splicing of exon 11, IR-A (without exon 11) and IR-B (with exon 11). Exon 11 is 36 bp and encodes 12 amino acids (717–729) in the COOH-terminus of the IR alpha-subunit. IR-A has higher binding affinity for insulin and IGF-II than IR-B. Interestingly, IR-A is predominantly expressed in fetal tissues, peripheral nerve, brain and tumours whilst IR-B is expressed primarily in classical insulin sensitive tissues such as adult liver and muscle. Our previous studies showed that in mice, like other species, the IR is expressed throughout preimplantation development. IR-B is expressed throughout the preimplantation period, whilst IR-A is expressed following compaction. Immunofluorescent confocal microsopy using an exon11 specific antiserum revealed IR-B immunoreactivity in cell membranes of zygotes and embryos to the morula stage and concentrated in the trophectoderm of blastocysts. Previous studies have shown that insulin can have proliferative effects prior to compaction.1 Consistent with a functional IR at the 2-cell stage, insulin treatment rapidly increased cytoplasmic staining for IR-B within 5-15 min suggesting IR internalization on binding of insulin, which may be either trafficking to the nucleus for regulation of transcription or bound for degradation. Further investigations are underway to address these two options. (1)Gardner, H. G., and Kaye, P. L. (1991). Insulin increase cell numbers and morphological development in mouse pre-implantation embryos in vitro. Reprod Fertil Dev 3, 79–91.

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