Low-speed purification of human placental nuclei

Reséndez‐Pérez, Diana; Barrera-Saldaña, Hugo A.; Morales-Vallarta, M.; Ramírez-Bon, Enrique; Leal-Garza, C. H.; Feria-Velazco, A.; Sánchez-Anzaldo, F. J.

doi:10.1016/s0143-4004(84)80006-5

Cited by 9 publications

(6 citation statements)

References 22 publications

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“…Unless otherwise indicated, cells were incubated overnight (16-18 h) in the same medium without FBS for all experiments. Cells harvested by scraping in ice-cold PBS (0.14 M NaCl͞8.1 mM Na 2 HPO 4 ͞1.5 mM KH 2 PO 4 ) (BioSource International, Camarillo, CA) were washed twice with the same solution before isolation, purification (17), and fractionation (18) of nuclei. Briefly, confluent cells from ten 15-cm plates (Ϸ3 ϫ 10 8 cells total) were sedimented by centrifugation at 1,000 ϫ g for 5 min and homogenized with 10 strokes in a 7-ml Dounce tissue grinder (Wheaton Scientific) in 4 ml of TKMS buffer (50 mM Tris, pH 7.5͞25 mM KCl͞5 mM MgCl 2 ͞250 mM sucrose) containing 0.5 mM 4-(2)-aminomethylbenzenesulfonyl fluoride, 10 g͞ml each leupeptin and aprotinin, and 1 g͞ml pepstatin A.…”

Section: Methodsmentioning

confidence: 99%

Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors

Padilla

Pacheco–Rodriguez

Moss

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an Ϸ200-kDa brefeldin A-inhibited guanine nucleotideexchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions.

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Section: Methodsmentioning

confidence: 99%

Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors

Padilla

Pacheco–Rodriguez

Moss

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Placental nuclei were prepared in two fractions by using the technique described by Resendez-Perez et al (18): nuclear fraction IV (NF-IV; ref. 18) containing knotted nuclei from syncytiotrophoblast and nuclear fraction III (NF-III) containing free nuclei from syncytiotrophoblast plus nuclei from other placental cell types as well as from contaminating maternal and fetal leukocytes.…”

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confidence: 99%

“…18) containing knotted nuclei from syncytiotrophoblast and nuclear fraction III (NF-III) containing free nuclei from syncytiotrophoblast plus nuclei from other placental cell types as well as from contaminating maternal and fetal leukocytes. For each placenta studied, nuclear fractions were prepared from :40 g of freshly obtained placental villous tissue.…”

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confidence: 99%

Detection of benzo[a]pyrene diol epoxide-DNA adducts in human placenta.

Manchester

Weston

Choi

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Human placenta is a readily available organ that responds to maternal environmental insult and has been previously used to investigate metabolism and bioactivation of procarcinogens, for example, benzo[alpyrene. HPLC in com-bination with synchronous fluorescence spectroscopy was used to examine 28 placentas for the presence of benzo[a]pyrene diol epoxide-DNA adducts, and 10 of these were found to be positive. DNA samples from these placentas were subsequently pooled and subjected to partial enzymatic digestion to oligonucleotide fragments. Concentration of those DNA fragments containing benzo[alpyrene diol epoxide-DNA adducts was achieved by immunoaffinity chromatography with polyclonal antibodies raised against these adducts. Column eluates were hydrolyzed under mild acid conditions and extracted with an organic solvent. The presence of benzo[a]pyrene-7,10/8,9-tetrahydrotetrol residues in the extracts was determined by HPLC and synchronous fluorescence spectroscopy and was confirmed by GC/MS. The results unequivocally confirm bioactivation and formation of DNA adducts from benzo[ajpyrene in human placenta in vivo and establish a methodological approach to direct measurement of carcinogen-DNA adducts that are formed as a result of human environmental exposure.

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“…Placentas from term, uncomplicated pregnancies were collected at delivery at University Hospital, Denver, according to a protocol approved by the University of Colorado Health Sciences Center Human Subjects Committee. For each placenta studied, nuclear fractions were prepared from approximately 40 g of freshly obtained placental villous tissue (syncytiotrophoblast tissue) (21). From these fractions DNA was purified to optical density absorbance ratios (OD 260 nm/280 nm) of 1.8 or greater by proteinase digestion, phenol extraction, ethanol precipitation, and RNase digestion according to a protocol developed to extract DNA from peripheral blood cells (19).…”

Section: Methodsmentioning

confidence: 99%

Derivative fluorescence spectral analysis of polycyclic aromatic hydrocarbon-DNA adducts in human placenta

Weston¹,

Manchester²,

Poirier³

et al. 1989

Chem. Res. Toxicol.

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Metabolic activation in humans of chemical carcinogens found in the environment results in the formation of carcinogen-DNA adducts in vivo. Some polycyclic aromatic hydrocarbon-DNA adducts in human DNA can be hydrolyzed under mildly acidic conditions to yield tetrahydrotetrol derivatives which may then be detected by synchronous fluorescence spectroscopy. In an analysis of human placental DNA, second derivative spectroscopy alone was unable to resolve the synchronous fluorescent signature for r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene from a crude extract, because a complex array of other fluorescent materials was also present. Purification of the sample by a combination of chromatographic procedures including immunoaffinity chromatography and HPLC has now been shown to yield r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene residues from human DNA that are spectroscopically pure at the second derivative level. Immunoaffinity columns were prepared with rabbit antiserum raised against DNA that had been modified with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyre ne. This antiserum has now been shown to recognize DNA samples that have been modified with six different polycyclic aromatic hydrocarbon diol epoxides and is probably only specific for a broad spectrum of polycyclic aromatic hydrocarbon-DNA adducts. Adducts were eluted from the immunoaffinity columns, hydrolyzed with acid, and extracted into isoamyl alcohol, before being subjected to high-performance liquid chromatography. These experiments reveal important limitations of second derivative fluorescence spectroscopy as a tool in the analysis of complex environmental mixtures. Furthermore, they extensively define the ability of anti-benzo[alpha]pyrenediol epoxide-DNA antibodies to recognize different types of polycyclic aromatic hydrocarbon-DNA adducts.

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Low-speed purification of human placental nuclei

Cited by 9 publications

References 22 publications

Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors

Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors

Detection of benzo[a]pyrene diol epoxide-DNA adducts in human placenta.

Derivative fluorescence spectral analysis of polycyclic aromatic hydrocarbon-DNA adducts in human placenta

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