SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The D protein (16 kDa) is part of a protein core, common to U1, U2, U5, U4/U6 small nuclear RNA containing ribonucleoprotein particles. Monoclonal antibodies reactive with the D protein were used in quantitative dot blotting and Western blotting to demonstrate that this protein was a component of salt resistant nuclear structures and was enriched greater than 3 to 5-fold in RNAase-protected nuclear matrix preparations.
The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrahymena into Xenopus cell-free extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assembled in vitro. Our previous works showed the 5'NTS (non-transcription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system, we found that the 5'NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xenopus eggs.
Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide (HMBA). Their nuclear matrix proteins (NMPs) were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.
We have identified DNA fragments which bind specifically to the nuclear matrix in vitro, termed matrix association regions (MARs), in the first and fourth introns of rat alpha 2-macroglobulin gene. The MAR in the first intron is enriched with sequences closely related to the cleavage consensus of topoisomerase II, and contains the binding site of nuclear factor-alpha, a sequence-specific DNA binding protein reported previously.
1. High molecular weight non-histone proteins (NHP) were isolated from Morris hepatoma 7777 by Sephadex G-100, S-200 chromatography. 2. Specific polyclonal antibodies were raised against these NHP in rabbits. These antibodies recognized specific NHP components present in Morris hepatoma 7777 and 8994, but not in normal rat liver. Hepatoma-associated antigens are phosphoproteins. 3. Immunologically specific NHP of Morris hepatoma are intensively concentrated in nuclear matrix fraction.
Nuclei assembled in vitro from Xenopus egg extracts and DNA display many properties known from in vivo nuclei. However, the distribution pattern of DNA in such nuclei remains unknown. We introduced rDNA from Tetrahymena macronuclei into Xenopus egg extracts and examined the association of specific DNA sequences with the nuclear matrix (NM) of the nuclei formed. We found that the 5' non-transcribed spacers (5'-NTS) specifically bound to the NM as previously shown in normal Tetrahymena macronuclei.
We evaluated the usefulness of urinary nuclear matrix protein 22 (NMP22) compared to urinary cytology in the detection of urothelial transitional cell carcinoma (TCC). Between July 1999 and March 2000, 227 patients complaining of microscopic or gross hematuria were analyzed. Twenty-four patients (10.6%) had urothelial TCC. The urinary NMP22 level was significantly higher in the patients with urinary TCC compared to the other patients. The sensitivity and specificity of the results obtained with urinary NMP22 were 58.3% and 84.2%, respectively, and those obtained by urinary cytology were 45.8% and 98.0%, respectively. False-positive results were obtained with urinary NMP22 in the patients with urinary diversion using intestine, bladder invasion from other cancers, urinary tract infection, and urolithiasis. The urinary NMP22 level was significantly associated with tumor stage, suggesting its usefulness for detection of urothelial TCC. However, although urinary NMP22 showed equal sensitivity for the detection of TCC, it was not superior to urinary cytology.
The clinical usefulness of the nuclear matrix protein 22 (NMP22) Bladder Chek test as a novel urine marker in the detection of patients with bladder cancer was evaluated in comparison with the urinary NMP22 enzyme-linked immunosorbent assay (ELISA) and urinary cytology. A total of 40 patients with pathologically proven bladder cancer voided urine specimen before treatment. The urine samples were divided for NMP22 Bladder Chek test, NMP22 ELISA, and urinary cytology. In the 40 patients with bladder cancer, the overall positive rate was 62.5% for the NMP22 Bladder Chek test, 55% for the NMP22 ELISA test, and 27.5% for urine cytology. There was a significant difference between NMP22 Bladder Chek, NMP22 ELISA and cytology. The positive rate with the NMP22 Bladder Chek and NMP22 ELISA was higher in the patients with high grade and large-size (1 cm < or =) tumor. In 40 patients presenting with microhematuria without urothelial cancer, the false positive rate 12.5, 10, and 0% for NMP22 Bladder Chek, NMP22 ELISA, and urinary cytology. No significant difference was found with the test. In conclusion, the urine NMP22 Bladder Chek test provided a higher positive rate than the NMP22 ELISA test and urinary cytology. Therefore, the NMP22 Bladder Chek test may be clinically more useful as a tumor marker for the diagnosis of bladder cancer.
After culture with 3H-thymidine murine erythroleukemia cells with more sites of DNA attached to the nuclear matrix have more labeled thymidine in their matrix-bound DNA than in their total DNA than do cells with fewer attachment sites. This indicates that the average attachment site sequence is enriched in A + T base pairs.
We analyzed the subcellular distribution of nuclear transport-defective simian virus 40 Lys-128-mutant (cT-3 [R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984] and d10 [D. Kalderon, W. D. Richardson, A. F. Markham, and A. E. Smith, Nature (London) 311:33-38, 1984]) large T antigens in various Lys-128-mutant-transformed rodent cells and in Lys-128-mutant d10-infected TC7 cells. Small but significant amounts of the mutant large T antigens were found in association with nuclear substructures, both in mutant-transformed and in mutant-infected cells. Experiments with TC7 cells made incompetent for cell division by 60Co irradiation supported the assumption that Lys-128-mutant large T antigen did not associate with nuclear components during mitosis but most likely was transported into the nucleus because the Lys-128 mutation was leaky for nuclear transport. Low-level simian virus 40 DNA replication and production of infectious mutant virus progeny in TC7 cells indicated that the association of Lys-128-mutant large T antigen with nuclear substructures is functional.
In chicken immature erythrocytes, approximately 4% of the modifiable histone lysine sites participate in active acetylation. There are two categories of actively acetylated histone H4. Although both are acetylated at the same rate (t1/2 = 12 min), one is acetylated to the tetraacetylated form and is rapidly deacetylated (class 1), and the other is acetylated to mono- and diacetylated forms and is slowly deacetylated (class 2). We show that the chromatin distribution of the class 1 labeled tetraacetylated H4 species paralleled that of the transcriptionally active DNA sequences. For example, the chromatin fragments of the insoluble nuclear material contained 76% of the active DNA and 74% of the labeled tetraacetylated H4. Class 2 labeled acetylated H4 species were found in repressed chromatin and were enriched in active/competent gene-enriched chromatin fragments. The majority of the histone deacetylase activity (75-80%) was located with the insoluble residual nuclear material. Further, approximately 40-50% of the enzyme activity was associated with nuclear matrices prepared by two methods using high salt and intermediate/high salt extraction. Histone deacetylase was solubilized by extracting the nuclear matrices with high salt and 2-mercaptoethanol, a procedure that generates nuclear pore-lamina complexes. These results demonstrate that histone deacetylase is a component of the internal nuclear matrix.
The immunoglobulin intragenic mu enhancer region acts as a locus control region that mediates transcriptional activation over large distances in germ line transformation assays. In transgenic mice, but not in transfected tissue culture cells, the activation of a variable region (V(H)) promoter by the mu enhancer is dependent on flanking nuclear matrix attachment regions (MARs). Here, we examine the effects of DNA methylation, which occurs in early mouse development, on the function of the mu enhancer and the MARs. We find that methylation of rearranged mu genes in vitro, before transfection, represses the ability of the mu enhancer to activate the V(H) promoter over the distance of 1.2 kb. However, methylation does not affect enhancer-mediated promoter activation over a distance of 150 bp. In methylated DNA templates, the mu enhancer alone induces only local chromatin remodeling, whereas in combination with MARs, the mu enhancer generates an extended domain of histone acetylation. These observations provide evidence that DNA methylation impairs the distance independence of enhancer function and thereby imposes a requirement for additional regulatory elements, such as MARs, which facilitate long-range chromatin remodeling.
Protein kinase CK2 is recognized as one of the key cellular signals for cell growth and proliferation. Its nuclear targeting appears to be critical to its role in these functions. In the nucleus, nuclear matrix (NM) which plays a major role in growth-related activities is a primary locus for CK2 signaling. A variety of growth stimuli evoke a rapid translocation of the CK2 to the NM whereas removal of these factors has the opposite effect. These studies, employing various experimental models of cell growth (involving different growth-stimulatory factors), have suggested that rapid shuttling of CK2 to the NM is a key feature of early growth control. By contrast, removal of growth-stimulatory factors leading to the loss of cell viability is associated with early loss of CK2 from the NM (and chromatin). This indicates that absence of CK2 from the nuclear compartment is contributory to induction of cell death via apoptosis, implying a protective role for CK2 against cell death. Here, we review the evidence that suggests that CK2 signaling in the NM is not only involved in cell growth but also in cell survival.
The Fas/Fas ligand system induces apoptosis, while soluble Fas (sFas) blocks the system and soluble Fas ligand (sFasL) functions to induce apoptosis. The assay of nuclear matrix protein (NMP) released from dead or dying cells can be used to quantitate cell death. Therefore, we evaluated the relationship among serum levels of NMP, sFas, and sFasL in patients with Graves' disease. We measured serum levels of sFas, sFasL, NMP, thyroid hormones and TSH receptor antibody in 20 normal control subjects (5 men, 15 women; mean age, 44.3 years), 32 patients with untreated Graves' disease (4 men, 28 women; mean age, 44.1 years), and 10 patients with Graves' disease treated by methimazole (3 men, 7 women; mean age 39.2 years). Serum NMP was significantly lower (10.4 +/- 4.3 IU/ml, p < 0.02) in patients with untreated Graves' disease than in patients with treated Graves' disease (16.4 +/- 7.3 IU/ml) and control subjects (15.3 +/- 8.9 IU/ml). Serum sFas and sFasL were significantly higher in patients with untreated Graves' disease than in patients with treated Graves' disease and in control subjects. In the patient groups with Graves' disease, serum NMP was negatively correlated with sFas (r = -0.612, p < 0.001) and serum sFas was positively correlated with FT4 (r = 0.360, p < 0.05) and TRAb (r = 0.384, p < 0.05). Serum NMP was correlated with sFas. These results suggest that serum NMP is decreased in patients with untreated Graves' disease, and that cell death or apoptosis in patients with Graves' disease is affected by soluble Fas under the influence of thyroid function.
Replication of the small DNA tumor virus, simian virus 40 (SV40), is largely dependent on host cell functions, because SV40, in addition to virion proteins, codes only for a few regulatory proteins, the most important one being the SV40 large tumor antigen (T-antigen). This renders SV40 an excellent tool for studying complex cellular and viral processes. In this review we summarize and discuss data providing evidence for virtually all major viral processes during the life cycle of SV40 from viral DNA replication to virion formation, being performed at or within structural systems of the nucleus, in particular the chromatin and the nuclear matrix. These data further support the concept that viral replication in the nucleus is structurally organized and demonstrate that viruses are excellent tools for analyzing the underlying cellular processes. The analysis of viral replication at nuclear structures might also provide a means for specifically interfering with viral processes without interfering with the corresponding cellular functions.
The phospholipid involvement in the regulation of the functional and structural properties of the nuclear matrix is discussed analysing the results obtained with the enzymic removal of these molecules. Namely phospholipids seem to mediate hydrophobic interactions between nucleic acids and matrix fibrils either directly or indirectly through an association with the non-histone proteins of the matrix.
The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.
Estradiol (E), diethylstilbestrol (DES), and three structurally similar DES derivatives and analogs, consisting of racemic mixtures of indenestrol-A (IA; rac), indenestrol-B (IB; rac), and the Z-isomer of pseudo-DES (PD) were used as probes to determine the effect of ligand on receptor-nuclear acceptor site interactions. IA (rac), IB (rac), and Z-PD have been previously shown to interact with the mouse uterine estrogen receptor with high affinity (Kd, approximately 1.5-2.2 x 10(-10) M), occupy similar levels of receptor in the nucleus in vivo, and have nuclear retention times similar to those of E and DES. However, in spite of these similarities, they differentially stimulate estrogenic responses that were previously thought to be interrelated and obligatory for full estrogenic action. In an attempt to elucidate the mechanism of differential stimulation of estrogen-regulated responses, the temporal pattern of KCl-resistant receptor sites was examined after a single injection of E, DES, IA (rac), IB (rac), Z-PD, or saline. Z-PD displayed lower levels of KCl-resistant receptor than the other compounds, whereas IA (rac) and IB (rac) had patterns similar to those of E and DES. In cell-free binding assays, all of the different receptor-ligand complexes were equally effective at competitively inhibiting the binding of receptor-[3H]E complexes to mouse uterine nuclear matrix with a Ki of 1.1 x 10(-10) M. Direct binding analysis showed no difference in the number of nuclear binding sites (range, 40.4-63.9 fmol/100 micrograms DNA) or Kd (range, 1.3-1.8 x 10(-10) M) among the different receptor-ligand complexes. Dissociation studies performed at 4 C showed no differences among the different complexes. In contrast, Z-PD-receptor complexes dissociated much faster from matrix sites at 22 C than any of the other complexes. Significant differences were noted in the proportion of complexes that were resistant to extraction with 0.6 M KCl. Forty-three percent of receptor-E complexes and 44% of receptor-DES complexes were resistant to KCl extraction, while 61% of receptor-IA and 29% of receptor-PD complexes were resistant to extraction, paralleling their activity in eliciting some estrogenic responses. In contrast, no difference was seen by gel shift assays in the ability of the ligand-receptor complexes to bind to a specific PS2 estrogen-responsive DNA sequence. These results demonstrate that the ligand may significantly affect the interaction of the estrogen receptor with nuclear acceptor sites.