79 PSEUDOPHYSIOLOGICAL TRANSCOMPLEMENTARY ACTIVATION OF GOAT OOCYTES RECEIVING ADULT EAR CUTANEOUS FIBROBLAST CELL NUCLEI

2010 ◽  
Vol 22 (1) ◽  
pp. 198
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Somatic Cell ◽  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Atmospheric Conditions ◽  
Cell Nuclei ◽  
Developmental Potential ◽  
Water Saturated

The aim of the study was to determine the in vitro developmental potential of caprine cloned embryos following pseudophysiological (transcytoplasmic) transcomplementary activation of oocytes reconstructed with ear skin-derived fibroblast cell nuclei. The source of nuclear recipient cells were IVM doe oocytes. The reconstruction of the previously enucleated oocytes (i.e. ooplasts) was performed by microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly into the cytoplasm. The reconstructed oocytes were incubated in Upgraded B2 INRA medium for 30 min to 1 h before their pseudophysiological activation. The activation was achieved by electrofusion of clonal cybrids with the allogeneic cytoplasts isolated from caprine IVF-created zygotes, which led to the formation of triple allocytoplasmic hybrids (allocybrids). These originate from 3 sources: (1) homologous whole nuclear donor fibroblast cells or their karyoplasts; (2) enucleated oocytes (ooplasts), and (3) zygote-derived cytoplasts. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The resulting zygoplast-clonal cybrid couplets were subsequently subjected to electrofusion, which was induced by application of a single DC pulse of 2.4 kV cm-1 for 15 μs. The electrofusion of zygoplast and reconstructed oocyte plasma membranes occurred in an isotonic dielectric solution deprived of Ca2+ ions. The transcytoplasmically activated clonal cybrids were cultured in vitro in Upgraded B2 INRA medium for 48 h at 38.5°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Afterward, cleaved embryos were co-cultured with Vero cells in medium supplemented with 10% fetal bovine serum for an additional 96 to 144 h up to morula and blastocyst stages under the same thermal and atmospheric conditions. A total of 53/78 (67.9%) oocytes reconstructed with fibroblast cell nuclei were successfully fused with zygoplasts. From among 53 cultured cloned embryos, 34 (64.2%) cleaved. The rates of embryos that reached the morula and blastocyst stages were 21/53 (39.6%) and 11/53 (20.8%), respectively. In conclusion, the relatively high percentages of morulae and blastocysts were noticed among in vitro-cultured caprine cloned embryos produced by the strategy of pseudophysiological transcytoplasmic activation of oocytes reconstructed with adult dermal fibroblast cell nuclei. Therefore, the use of cytoplasmic components originating from zygotes as the stimuli for activation of nuclear-transferred oocytes appeared to be an effective procedure in the generation of goat blastocysts by somatic cell cloning.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
W. Mlodawska ◽  
J. Kochan ◽  
A. Okolski ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Contact Inhibition ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Condensed Chromosome ◽  
Donor Cells

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 217-222 ◽  
Author(s):  
Xi Jun Yin ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Cell Number ◽  
Heart Beat ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Average Cell ◽  
Developmental Potential ◽  
Membrane Protrusions ◽  
Implantation Sites

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.

scholarly journals Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intravitam for apoptosis occurrence / Uzyskiwanie klonalnych zarodków świni z wykorzystaniem komórek fibroblastycznych poddanych inhibicji kontaktowej lub deprywacji troficznej oraz analizowanych przyżyciowo w kierunku apoptozy

2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela
Keyword(s):  
Dermal Fibroblast ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Donor Cells ◽  
Cloned Pig

Abstract Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.

scholarly journals Assessment of in Vitro Developmental Capacity of Porcine Nuclear-Transferred Embryos Reconstituted with Cumulus Oophorus Cells Undergoing Vital Diagnostics for Apoptosis Detection / Ocena zdolności rozwojowych in vitro klonalnych zarodków świni rekonstytuowanych z jąder komórek wzgórka jajonośnego poddawanych przyżyciowej diagnostyce w kierunku wykrywania apoptozy

2013 ◽  
Vol 13 (3) ◽  
pp. 513-529 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska
Keyword(s):  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Annexin V ◽  
Cumulus Cells ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Cumulus Oophorus ◽  
Group I ◽  
Cloned Pig

Abstract The objective of the current investigation was to extensively compare the in vitro developmental capabilities between cloned pig embryos reconstructed with the cell nuclei of either cumulus oophorus cells or adult dermal fibroblast cells that were both evaluated as non-apoptotic on the basis of YO-PRO-1- and Annexin V-eGFP-mediated vital analysis for programmed cell suicide. In Group I, the competences of nuclear-transferred (NT) embryos that were derived from non-apoptotic/ non-necrotic (i.e., YO-PRO-1- and Annexin V-eGFP-negative) cumulus cells to complete their development to the morula and blastocyst stages were maintained at the proportions of 155/364 (42.6%) and 54/364 (14.8%), respectively. In Group II, NT embryos that were reconstituted with non-apoptotic and/or non-necrotic adult cutaneous fibroblast cells developed to the morula and blastocyst stages at the rates of 207/358 (57.8%) and 110/358 (30.7%), respectively. Although the in vitro developmental potential of porcine NT embryos derived from non-apoptotic/non-necrotic cumulus cells was significantly lower (P<0.001) than that of NT embryos reconstructed with adult dermal fibroblast cells, the obtained morula/blastocyst formation rates turned out to be considerably higher as compared to the rates reported by other investigators. Altogether, to our knowledge, the comprehensive research aimed at the determination of preimplantation developmental outcomes of cloned pig embryos produced using nuclear donor somatic cells of different provenance (cumulus oophorus cells or adult cutaneous fibroblast cells) that were vitally diagnosed for the lack of proapoptotic transformations in their plasma membranes has not yet been accomplished.

76 DEVELOPMENTAL CAPABILITY OF TRANSGENIC NUCLEAR-TRANSFERRED PIG EMBRYOS PRODUCED USING THE NOVEL METHOD OF PSEUDOPHYSIOLOGICAL ACTIVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 196 ◽  
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
R. Slomski
Keyword(s):  
Somatic Cell ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Original Method ◽  
Calcium Oscillations ◽  
Cell Nuclei ◽  
Novel Method ◽  
Transgenic Embryos

The physicochemical stimuli, which are commonly used for artificial activation of porcine nuclear-transferred (NT) oocytes, can affect detrimentally or cytotoxically the clonal cybrids and thereby inhibit the development or decrease the quality of cloned embryos. Therefore, we have recently developed a novel method of pseudophysiological transcomplementary (transcytoplasmic) activation to stimulate the developmental program of porcine oocytes reconstructed by somatic cell nuclear transfer. The mechanism underlying this original technique of activation is transcytoplasmic influx of sperm-derived proteins triggering intracellular calcium oscillations, which is mediated via heterologous (rabbit) zygote-descended cytoplasts. The purpose of our study was to estimate the in vitro developmental competences of porcine transgenic cloned embryos following pseudophysiological activation of oocytes receiving pWAPhGH-GFPBsd gene construct-nucleofected fetal fibroblast cell nuclei. In the cloning procedure, IVM pig oocytes were used as recipient cells for cell nuclei of positively selected transgenic fibroblast cells. The reconstruction of enucleated oocytes was performed by intracytoplasmic injection of either the somatic cell-derived karyoplast or whole tiny nuclear donor cell. The activation of porcine NT oocytes was achieved by electrofusion of them with the xenogeneic cytoplasts isolated from in vivo-derived rabbit zygotes (i.e. with the so-called zygoplasts), which led to the formation of triple xenocytoplasmic hybrids (xenocybrids). The rabbit zygotes had been flushed postmortem from the separated oviducts of superovulated postpubertal female donors 18 to 20 hafter administration of hCG and copulation. Single rabbit zygote-descended cytoplasts were inserted into the perivitelline space of previously reconstructed pig oocytes. The resulting zygoplast-NT oocyte couplets underwent fusion, which was induced by generation of 2 successive DC pulses of 1.2 kV cm-1 for 60 μs. The electrofusion medium consisted of 0.3 M Ca2+-deprived mannitol supplemented with 0.1 mM MgSO4 and 0.2 mg mL-1 fatty-acid-free BSA. The transcytoplasmically activated xenocybrids were cultured in vitro for 6 to 7 days up to morula/blastocyst stages. A total of 183/207 (88.4%) oocytes reconstructed with nucleofected fibroblast cell nuclei were successfully fused with zygoplasts. Out of 183 cultured NT embryos, 138 (75.4%) were cleaved. The rates of transgenic NT embryos that reached the morula and blastocyst stages yielded 106/183 (57.9%) and 65/183 (35.5%), respectively. In conclusion, the original method of pseudophysiological activation of porcine NT oocytes turned out to be relatively efficient, which has been confirmed by the high percentages of pWAPhGH-GFPBsd transgenic embryos developing in vitro to morula and blastocyst stages.

The effect of the time interval between injection and parthenogenetic activation on the spindle formation and the in vitro developmental potential of somatic cell nuclear-transferred rat oocytes

Zygote ◽  
2009 ◽  
Vol 18 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Shigetoshi Mizumoto ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Time Interval ◽  
Cell Nuclei ◽  
Spindle Formation ◽  
Developmental Potential ◽  
Developmental Rates ◽  
Culture Duration ◽  
Oocyte Cytoplasm ◽  
Preincubation Time

SummaryWe examined the optimal conditions for somatic cell nuclear transfer (SCNT) in the rat. First, we examined the effect of preincubation time before activation on SCNT rat oocytes produced in the presence of MG132 with regard to spindle formation and the potential to develop into blastocysts. The spindles of SCNT oocytes continued to elongate with an increase in the culture duration and, in approximately half of oocytes, the chromosomes were distributed along the spindles at 120 min after incubation. Such abnormal spindle formation in SCNT oocytes is a possible reason for the low developmental potential of SCNT rat oocytes. To inhibit the formation of abnormal spindle formation, we examined secondly the developmental potential of rat SCNT oocytes that had been preincubated with nocodazole and demecolcine instead of MG132. The developmental rates in SCNT oocytes, however, were decreased. For successful rat somatic cell cloning, two steps might be required: (1) to culture the somatic cell nuclei for a sufficient time in MII oocyte cytoplasm to enhance nuclear reprogramming; and (2) to induce normal spindle formation with normal chromosomal construction.

scholarly journals Developmental ability of cloned embryos from neural stem cells

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 849-857 ◽  
Author(s):  
Eiji Mizutani ◽  
Hiroshi Ohta ◽  
Satoshi Kishigami ◽  
Nguyen Van Thuan ◽  
Takafusa Hikichi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Success Rate ◽  
Sertoli Cells ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cloning Efficiency ◽  
Cell Nuclei ◽  
Developmental Potential ◽  
Undifferentiated State

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2–3.5%). Although thein vitrodevelopmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula–blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point forin vitrodevelopment of cloned embryos depends on the donor cell type.

scholarly journals Tribological, biocompatibility, and antibiofilm properties of tungsten–germanium coating using magnetron sputtering

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mustafa Şükrü Kurt ◽  
Mehmet Enes Arslan ◽  
Ayşenur Yazici ◽  
İlkan Mudu ◽  
Elif Arslan
Keyword(s):  
Cell Culture ◽  
Magnetron Sputtering ◽  
Cell Line ◽  
Borosilicate Glass ◽  
Chromosomal Abnormalities ◽  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Antibiofilm Activity ◽  
Borosilicate Glasses

AbstractIn this study, borosilicate glass and 316 L stainless steel were coated with germanium (Ge) and tungsten (W) metals using the Magnetron Sputtering System. Surface structural, mechanical, and tribological properties of uncoated and coated samples were examined using SEM, X-ray diffraction (XRD), energy-dispersive spectroscopy, and tribometer. The XRD results showed that WGe2 chemical compound observed in (110) crystalline phase and exhibited a dense structure. According to the tribological analyses, the adhesion strength of the coated deposition on 316 L was obtained 32.8 N, and the mean coefficient of friction was around 0.3. Biocompatibility studies of coated metallic biomaterials were analyzed on fibroblast cell culture (Primary Dermal Fibroblast; Normal, Human, Adult (HDFa)) in vitro. Hoescht 33258 fluorescent staining was performed to investigate the cellular density and chromosomal abnormalities of the HDFa cell line on the borosilicate glasses coated with germanium–tungsten (W–Ge). Cell viabilities of HDFa cell line on each surface (W–Ge coated borosilicate glass, uncoated borosilicate glass, and cell culture plate surface) were analyzed by using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay. The antibiofilm activity of W–Ge coated borosilicate glass showed a significant reduction effect on Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) adherence compared to control groups. In the light of findings, tungsten and germanium, which are some of the most common industrial materials, were investigated as biocompatible and antimicrobial surface coatings and recommended as bio-implant materials for the first time.

Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

1995 ◽  
Vol 108 (6) ◽  
pp. 2187-2196 ◽  
Author(s):  
L.J. Wangh ◽  
D. DeGrace ◽  
J.A. Sanchez ◽  
A. Gold ◽  
Y. Yeghiazarians ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Nuclear Transplantation ◽  
Optimal Time ◽  
Genome Replication ◽  
Cell Nuclei ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 × 10(9) base pairs of DNA/per nucleus are synthesized in 30–40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.

Sodium affinity of brain Na(+)-K(+)-ATPase is dependent on isozyme and environment of the pump

1990 ◽  
Vol 258 (5) ◽  
pp. C803-C811 ◽  
Author(s):  
J. L. Brodsky ◽  
G. Guidotti
Keyword(s):  
Plasma Membranes ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Synaptic Plasma Membranes ◽  
Apparent Dissociation Constant ◽  
Low Sodium ◽  
Physiological Relevance ◽  
Mouse Fibroblast Cell Line

The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.

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