Increase In Nuclear Protein Research Articles

Dynamics of DNA–Protein Complex Formation in Rat Liver during Induction by Phenobarbital and Triphenyldioxane

CYP2B gene expression in liver of rats treated with phenobarbital and triphenyldioxane at early stage of induction (40 min-18 h) was studied using electrophoretic mobility shift assay (EMSA) and RT-PCR. During first 6 h after induction, differences in the dynamics of formation of DNA-protein complexes were shown for each inducer. Later (18 h after induction), the intensity pattern of these complexes became the same for both phenobarbital and triphenyldioxane treated animals. This suggests the existence of specific signaling for each inducer only in early stages of CYP2B activation. Increase in nuclear protein (possible transcription factor) binding to Barbie-box regulatory sequence of CYP2B genes was accompanied by their increased expression. Thus, we have demonstrated for the first time that early stages of induction (40 min and 3 h after administration of phenobarbital and triphenyldioxane, respectively) are accompanied by activation of nuclear proteins that can bind to Barbie-box element of CYP2B. Although various chemical inducers cause distinct activation of such binding, this process involves activation of gene transcription.

Activation of Nuclear Factor κB in Single Living Cells: DEPENDENCE OF NUCLEAR TRANSLOCATION AND ANTI-APOPTOTIC FUNCTION ON EGFPRELA CONCENTRATION

We have studied the dynamics of nuclear translocation during nuclear factor kappaB activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Quantitation of expression levels indicates that EGFPRELA can be detected at physiological concentrations of about 60,000 molecules per cell. Stimulation of transfected fibroblasts with interleukin (IL)-1beta caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomitant 20% decrease in cytoplasmic levels. The response of individual cells to IL-1beta was graded, and the kinetics of nuclear translocation were dependent on the dose of IL-1beta and the level of EGFPRELA expression. The rate of nuclear uptake was saturable, and the time lag for uptake increased at higher EGFPRELA expression levels. Furthermore, nuclear translocation was reduced at less than saturating doses of IL-1beta suggesting that the pathway is limited by incoming signals. The response to IL-1beta was biphasic, demonstrating a decline in nuclear import rate at expression levels above three to four times endogenous. This correlated with the anti-apoptotic function of EGFPRELA which was more prominent at low expression levels and demonstrated successively less protection at higher levels. In comparison, transfection of p50 had no effect on the level of apoptosis and demonstrated some toxicity in combination with EGFPRELA.

CAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products.

Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with p(delta)ACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of p(delta)ACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, beta-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21ras. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.

Activator protein-1 (AP-1) is stimulated by microbial superantigens in human monocytic cells.

Microbial superantigens bind to major histocompatibility complex (MHC) class II molecules and activate gene transcription in monocytes. In search of transcription factors that potentially mediate the effects of superantigens at the nuclear level, we examined the capacity of staphylococcal superantigens to stimulate the activity of the transcriptional promoter factor activator protein-1 (AP-1). Electrophoretic mobility shift assays showed an increase in nuclear proteins that bound to the consensus AP-1 motif within 5 min following the stimulation of the monocytic cell line THP-1 with toxic shock syndrome toxin-1 (TSST-1) or staphylococcal endotoxin A. We show that mRNA levels for the subunits that compose AP-1, the protooncogenes c-fos and c-jun, are upregulated by stimulation of THP-1 cells with TSST-1. The activated AP-1 complexes were functional, as evidenced by the capacity of TSST-1 to stimulate the expression of an AP-1-driven reporter gene construct transfected into THP-1 cells. These results establish that the engagement of MHC class II molecules by superantigens increases the activity of functional AP-1 complexes and that this may proceed in part by transcriptional activation of c-fos and c-jun protooncogenes.

Apparent alterations in the early stage of excision repair of UV-induced DNA damages in a HeLa mutant cell line that is resistant to genotoxic stresses

We have previously reported a cisplatin-resistant HeLa cell line featuring a cross-resistance to genotoxic stresses including ultraviolet (UV) radiation and an enhancement of plasmi reactivation. In this study, excision repair of UV-DNA adducts in this resistant cell line was investigated. Using alkaline elution analysis, this resistant cell line showed a 2-fold enhancement in damage incision-associated DNA strand breaks. Using a gel mobility shift assay, the resistant cells exhibited a 3-fold increase in nuclear proteins which specifically recognize UV-damaged DNA. However, the rate of repair synthesis in the resistant cells appeared to be the same as in their parental counterparts. Thus, recognition and incision activities, the early stage of excision repair, are altered in the resistant cells. The result suggest that phenotypic cross-resistance of this cell line to UV is probably due to an improved excision repair of UV-DNA adducts which is defective in xeroderma pigmentosum group A cells. The results are consistent with the conclusion that the early stage, including recognition and incision, of excision repair is critical in determining cellular sensitivity of resistance to DNA damage.

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.

Removal of excess nuclear protein from cells heated in different physiological states

The relationship between heat-induced cell kill and alterations in nuclear protein content was investigated by heating quiescent 66 mouse mammary adenocarcinoma cells in three different physiological states: (1) quiescent, nutrient deprived cells (Q); (2) Q cells placed in fresh medium 2 h prior to heating (QM); and (3) Q cells made thermotolerant by a previous heat treatment (QTT). Although cell survival varied by a factor of 80, the increase in nuclear protein after a 30 min exposure to 45 degrees C was similar in Q, QM and QTT cells. Removal of the excess nuclear protein from cells in the three physiological states differed both in the percentage of the population that could remove the protein and the rate at which the protein was removed. While all of the QM and QTT cells removed the excess nuclear protein, approximately 30% of the Q cells did not remove the excess nuclear protein, and continued to accumulate protein over the 48 h after the heat treatment. The time for complete removal of the excess protein (Q, 32 h: QM, 18 h: QTT, 8 h) was directly correlated with cell survival. Therefore, these data support the hypothesis that the removal of excess nuclear protein after heat treatment is related to and, perhaps, a determinant of, cell survival.

Age-related alterations in human chromosome composition and DNA content in vitro during senescence.

SummaryDifferent theories of ageing involving somatic mutations, error catastrophe, compensation and repair, and programmed ageing were subjected to analysis for their feasibility from experimental data. Due to the relative difficulty of carrying out longitudinal studies in human subjects in vivo and the long periods involved, most of these experiments dealt with in vitro systems.I. Fibroblast cells in culture were found to be ideal materials for demonstrating senescence for a particular species and at the terminal end the cultures showed certain changes associated with age.II. It appears that the ideas regarding the alterations in DNA content at the terminal stages of the culture or otherwise are related to the tissues concerned and the duration of the cultured condition.III. The importance of DNA content specially related to the concept of stability of the DNA strand supports indirectly the error catastrophe theories. The rate of net DNA synthesis, the size of the replicon and duration of S phase is reported not to change during in vitro ageing. The regulation system of DNA replication may however change, but this alteration does not affect the DNA replication machinery. Following cell fusion studies it has been hypothesized that senescent human diploid fibroblasts contain a diffusible inhibitor which blocks cells at G1 phase. Some immortal cell lines like HeLa and SV40‐transformed cells would contain a dominant inducer that could override or inactivate the putative inhibitor, the nature of which is not yet clear.IV. DNA repair competence of fibroblast cultures is reported to decline near the end of in vitro life‐span. However, it has been noted that the human skin fibroblasts from both young and old donors are equally proficient in repairing damage by UV light.V. The replication patterns of chromosomes from both senescent embryonic fibroblasts and early‐passage adult skin fibroblasts were essentially identical. There were very few differences between the early‐passage embryonic and adult skin cells. It was concluded that the terminal replication pattern of fibroblasts changes very little with cellular ageing.VI. A statistically significant increase in sister chromatid exchange frequency has been reported during the terminal part of fibroblast cultures.VII. Electron‐microscopic studies have confirmed the increased organization of microfillaments into bundles in senescent cells. The presence of a rigid cytoskeletal structure may contribute in part to the inability of such cells to replicate.VIII. Age‐related increase in nuclear proteins was attributed to accumulation of residual acid proteins. Densitometric analysis showed that histone HI was low in late‐passage cells and H4 fraction increased relatively at the terminal phase.IX. In contrast to age‐matched controls, fibroblasts cultured from progeria and Werner's syndrome undergo significantly low population doubling. Metaphase plates from these patients demonstrated a much higher frequency of chromosomal abnor malities than normal fibroblasts. Frequency of sister chromatid exchange in cells from Fanconi's anaemia did not show any significant change as compared with control sets.X. Significantly lower Feulgen DNA values have been recorded from lymphocytes of the elderly as compared with younger ones, indicated by hypodiploidy as well as by the individual amounts of euchromatin and heterochromatin. However, later data from flow‐cytometric measurements indicated that DNA content was the same for all age groups.XI. The UV‐induced unscheduled DNA synthesis in lymphocytes of 80 to 90 year‐old individuals was reduced as compared to younger persons. However, the rate of repair of DNA strand breaks is apparently constant in all the age groups.XII. Increase in aneuploidy from lymphocyte cultures of aged individuals has been recorded by many workers.XIII. Significantly lower titres of serine, threonine, histidine, ornithine and lysine have been observed in aged persons, and only the first three decreased equally in both sexes. Some of the amino acids were influenced by sex hormones.XIV. Study of the frequency of spontaneous sister chromatid exchanges showed that neither intra‐individual variation between replicate cultures established from the same blood sample nor variation among samples from the same individual initiated at different times was significant. However, sensitivity to induced sister chromatid exchange is reported to increase with age.

Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constituents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.

Role of RNA and Protein Synthesis and Turnover in the Heat-Induced Increase in Nuclear Protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.

Two-dimensional gel analysis of nuclear proteins during muscle differentiation in vitro: I. Changes in nuclear protein content

Improved methods are described for the isolation of nuclei from skeletal muscle-cell cultures by lysis with Triton X-100 and centrifugation through 65% sucrose-5 mM MgCl 2. Evidence is presented that levels of contamination by cytoplasmic and fibrillar proteins are acceptably low. Nuclear proteins were separated into saline-soluble and non-histone protein (NHP) fractions and analysed on two-dimensional gels stained with Coomassie Blue. 2D-gels of NHP and cytoplasmic proteins were compared and few proteins common to both fractions were found. Of three major cytoplasmic proteins, actin, tubulin and tropomyosin, only actin could be positively identified as a minor protein in NHP and tropomyosin was undetectable. An overall increase in nuclear NHP content (relative to DNA) occurred during the early period of cell differentiation in vitro (24–66 h of culture) and a corresponding increase in nuclear protein to DNA ratio was observed. There were differential changes between individual NHP, some major proteins showing little change, while others showed large increases. These changes may be related to increases in nuclear protein uptake which are reported to occur during differentiation and hormone-induced gene transcription in other cell types.

Age-related decline in histone H1 fraction in human diploid fibroblast cultures

The age-related increase in cell volume and nuclear size of cultured human diploid fibroblasts reflected the accumulation of proteins in cytoplasm and nuclei of growth-retarded fibroblasts. Determination of the amount of nuclear proteins, which were fractionated into 0.15 M NaCl-soluble proteins, 0.4 N H 2SO 4-extractable proteins and residual acidic proteins, indicated that age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins. However, electrophoretic fractionation of histones from various passages of fibroblast cultures on acid urea polyacrylamide gel revealed that the relative amount of H1 fraction decreased with in vitro aging. This was further confirmed by mixing experiments examining the distribution of radioactivity of the histones from cell mixtures of young and senescent cultures labeled with [ 3H]lysine or [ 14C]lysine. A pulse label and chase experiment indicated that the observed decrease in the amount of histone H1 was mainly due to decrease in synthesis of histone H1 in senescent human fibroblast cultures.

Two-Dimensional Electrophoretic Analysis of Nuclear Acidic Proteins in Senescent Human Diploid Cells

Nuclear acidic proteins solubilized from young and senescent human diploid fibroblasts were analyzed by two-dimensional gel electrophoresis. The age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins. Differences in seven major nuclear acidic proteins between young and senescent cells were observed. [35S]-menthionine autoradiography showed that senescent cells had lost the ability to synthesize detectable amounts of four major proteins that are found in young cells. In addition, senescent cells synthesized two new major proteins that were undetectable in young cells. The isoelectric point of a single polypeptide with the molecular weight 37, 000 seemed to shift from 5.3 to 5.8 with cellular aging.

Initiation, Growth and Nuclear Characteristics of Tissue Cultures of Paeonia suffruticosa

AbstractTissue cultures of the garden paeony, Paeonia suffruticosa have been established using explants of etiolated stems. Callus formation was induced on agar‐solidified media containing ammonium ions or amino acids together with the hormones 2,4‐dichlorophenoxyacetic acid and kinetin, but not on media lacking the reduced nitrogen component. Attempts to induce callus from explants from green plants were completely unsuccessful and were characterized by the production of intense brown colorations, both of the explant and the medium. Subcultured tissue without the added hormones produced roots, both on solid and liquid media.Growth was tested on a range of liquid media, SH/2, SH, SH × 2 and SH—M, containing 1250, 2500, 5000 and 2500 mg/l potassium nitrate. The SH—M medium also contained 1650 mg/l ammonium nitrate. Growth measured as increased fresh weight was best in the SH/2, SH and SH—M media and was curtailed in the SH × 2 medium. Soluble protein content was highest at the lowest nitrogen concentration.A histochemical comparison of tissue grown on the SH/2, SH—M and SH—M lacking hormones showed that the cells in all the cultures remained diploid while differences were established in total nuclear protein, measured using the ninhydrin‐Schiff procedure. Nuclei from SH—M grown cells have a higher protein content than those from the SH/2 medium while cells from the SH—M medium lacking hormones show a further increase in nuclear protein. This raises the question whether this change in nuclear protein is related to the morphogenesis of roots which occurs in this medium.