Intranuclear Proteins Research Articles

Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. Here, we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix. We discuss this hypothesis in the context of recent observations from phylogenetically diverse organisms that nuclear envelope and intranuclear proteins form a highly dynamic and malleable structure that contributes to mitotic spindle function. We suggest that the viscoelastic properties of such a matrix may constrain spindle length while at the same time facilitating microtubule growth and dynamics as well as chromosome movement. A corollary to this hypothesis is that a key determinant of spindle size may be the amount of nuclear proteins available to form the spindle matrix. Such a matrix could also serve as a spatial regulator of spindle assembly checkpoint proteins during open and semi-open mitosis.

Nuclear envelope and lamin B2 function in the central nervous system

The nuclear envelope (NE) embraces the genome with a rich tapestry of outer and inner membrane proteins, some of which mechanically link the cytoskeleton to the nucleoskeleton (1). The nucleoskeleton includes the nuclear intermediate filaments, formed by independent networks of A- and B-type lamins. Humans have one gene (LMNA) encoding A-type lamins and two genes encoding B-type lamins (LMNB1 and LMNB2) (2). Fascinating links to human physiology have emerged as mutations in LMNA were found to cause a spectrum of disorders, generally termed “laminopathies.” Most known laminopathies affect one or more specific “load-bearing” tissues, with clinical phenotypes that include muscular dystrophy, cardiomyopathy, skin or bone defects, or widespread connective tissue defects that lead to segmental progeroid (“accelerated aging”) syndromes (3). Certain mutations in LMNA can even cause lipodystrophy and diabetes, or Charcot-Marie-Tooth axonal neuropathy. However, the central nervous system was conspicuously and puzzlingly unaffected, even in LMNA knockout mice. A new report in a recent issue of PNAS (4) fills this major gap in knowledge by showing that lamin B2 knockout mice have grossly abnormal development of the cerebral cortex and cerebellum. Lamins A, B1, and B2 show complex and differential patterns of expression in developing neurogenic regions of the rat brain (5). Lamins B1 and B2 are each expressed at the highest levels in different neuronal cell types, suggesting both overlapping and potentially unique roles during brain development (5). Neuronal functions for B-type lamins were first seen in Drosophila, which has one A-type lamin and one B-type lamin; mutating the B-type lamin disrupted the developmentally regulated migration of nuclei within photoreceptor cells (6). Mice deficient in lamin B1 die soon after birth with multiple phenotypes including lung and bone defects (7). The new study by Coffinier et al. (4) shows that lamin B2 knockout mice have severe defects in the layering of neurons in the cerebral cortex and cerebellum. This phenotype, known as lissencephaly, is due to defective migration of nuclei within developing lamin B2-deficient mouse neurons. Thus, lamin B2 is a crucial nucleoskeletal component of the nuclear migration machinery in mammals, along with the NE-spanning LINC complexes formed by SUN-domain proteins and Syne/Nesprin proteins (8, 9). Lamin B2 now provides the molecular “platform” needed for researchers to identify the other NE and intranuclear proteins that interact functionally with lamin B2 during nuclear migration, and map new genes relevant to lissencephaly.

111In-Labeled Immunoconjugates (ICs) Bispecific for the Epidermal Growth Factor Receptor (EGFR) and Cyclin-Dependent Kinase Inhibitor, p27Kip1

Probing intranuclear proteins in breast cancer (BC) cells by using radiolabeled antibodies is restricted by delivery barriers presented by cell and nuclear membranes. Our aim was to construct immunoconjugates (ICs) bispecific for epidermal growth factor receptors (EGFRs) and the intranuclear cyclin-dependent kinase inhibitor (CDKI) p27(Kip1) modified with nuclear-localizing sequences (NLSs) to facilitate their nuclear uptake following EGFR-mediated internalization. Bispecific ICs were constructed by first modifying EGF with peptides [GGPKKKRKVGYGCG] harboring NLS from SV-40 large T-antigen (underlined), then conjugating NLS-EGF to anti-p27(Kip1) antibodies through an extended PEO(12)-maleimide linker (Compound 1). Analogous ICs were constructed by using mouse IgG (Compound 2), a disrupted NLS (Compound 3) or omission of the EGF moiety (Compound 4). Binding to EGFR on MDA-MB-468, H2N, or HR2 BC cells and to p27(Kip1) in HELA cell lysate was measured. Internalization and nuclear importation were evaluated. Retention of the ICs in H2N or trastuzumab (Herceptin)-resistant HR2 cells exposed to trastuzumab to modulate p27(Kip1) expression with/without coexposure to the IGF-1 receptor kinase inhibitor, AG1024, was determined. Trastuzumab (10 microg/mL) unexpectedly decreased p27(Kip1) expression by 1.7-2.4-fold in H2N or HR2 cells. Conjugation of EGF to anti-p27(Kip1) antibodies (Compound 1) decreased the binding affinity of the ICs 7-fold toward EGFR and p27(Kip1). All ICs bound EGFR on MDA-MB-468 cells except Compound 4. Compound 1 was internalized into H2N cells over 48 hours and Compound 2 exhibited 1.6-fold greater nuclear importation in H2N or MDA-MB-468 cells than Compound 3. There was a significantly lower retention of Compound 1 in H2N cells exposed to trastuzumab, compared to unexposed cells, corresponding to decreased p27(Kip1), but in HR2 cells, diminished retention was observed only when these cells were coexposed to trastuzumab and AG]024. We conclude that (111)In-labeled bispecific ICs were constructed that specifically bound EGFR and p27(Kip1). These ICs were internalized into BC cells expressing EGFR and HER2 and imported into the nucleus. Their decreased retention by cells with trastuzumab-modulated p27(Kip1) suggests that they may be useful for probing this CDKI by imaging.

Update:Peptide Motifs for Insertion of Radiolabeled Biomolecules into Cells and Routing to the Nucleus for Cancer Imaging or Radiotherapeutic Applications

Intracellular compartments, in particular the cytoplasm or nucleus, have generally been poorly accessible or inaccessible to radiolabeled biomolecules (e.g., monoclonal antibodies [mAbs], peptides, or oligonucleotides [ODNs]). However, recently cell-penetrating peptides (CPPs) and nuclear localizing peptide sequences (NLSs) have been shown to have the capability of inserting biomolecules into cells and transporting them to the cell nucleus. This discovery now presents intriguing new opportunities to design radiopharmaceuticals that could potentially probe, through imaging, the expression of key intracellular or intranuclear regulatory proteins that define the tumor phenotype, predict outcome, or act as sensitive reporters of response or resistance to treatment. CPPs could also more efficiently internalize radiolabeled antisense ODNs or peptide nucleic acids (PNAs) into tumor cells to enhance the sensitivity of imaging gene expression at the mRNA level. Perhaps one of the most exciting new developments to emerge is the use of NLS to route mAbs and peptides conjugated to nanometer-micrometer range Auger-electron-emitting radionuclides (e.g., (111)In) to the nucleus of cancer cells following their receptor-mediated internalization. In the nucleus, these electrons are highly potent in causing lethal DNA strand breaks. In some cases, NLSs are present naturally in peptide growth factors or their receptors, where they function to deliver internalized ligands to the nucleus, or alternatively, they can be introduced synthetically. This update reviews the properties of CPPs and NLS and focuses on their use for inserting radiolabeled biomolecules into cancer cells for imaging or targeted Auger electron radiotherapy of malignancies.

Progesterone receptors in the human uterus and their possible role in parturition

An overview is given on the role of progesterone in parturition in the human. Progesterone withdrawal is considered to be a major event for the beginning of parturition. However, in the human, no evidence exists in favour of a decline in placental progesterone production prior to labour. Progesterone actions are mediated by two functionally different but structurally highly related intranuclear proteins, progesterone receptor (PR) A and PRB. In the human, functional progesterone withdrawal is thought to play a role. This may be mediated by a change in the expression of the two isoforms of the PR, with an increase in the PRA:PRB ratio, and this is accompanied by an increase in the expression of the estrogen receptor. These mechanisms are considered to be critical for the endocrine control of parturition.

XMAN1, an inner nuclear membrane protein, antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos.

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.

Nuclear Lipid Signaling

Abundant evidence now supports the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope as part of a bilayer membrane, but are actually within the nucleus in the form of proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet entirely clear. Generation of DAG within the nucleus is believed to recruit protein kinase C (PKC) to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P3 may mobilize Ca2+ from the space between the nuclear membranes and thus increase nucleoplasmic Ca2+. Less well understood are the increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus, (i) two routes of synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; (ii) two sources of DAG, one from the PI-PLC pathway and the other probably from phosphatidylcholine; (iii) several isoforms of PKC translocating to nuclei; (iv) increases in activity of the PI-PLC pathway at two points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P3, which may have several functions, including a role in the transfer of mRNA out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and, in particular, 3-phosphorylated inositol lipids, which are now emerging as possible major players in nuclear signaling.

Nuclear lipid signaling.

Abundant evidence now supports the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope as part of a bilayer membrane, but are actually within the nucleus in the form of proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet entirely clear. Generation of DAG within the nucleus is believed to recruit protein kinase C (PKC) to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P3 may mobilize Ca2+ from the space between the nuclear membranes and thus increase nucleoplasmic Ca2+. Less well understood are the increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus, (i) two routes of synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; (ii) two sources of DAG, one from the PI-PLC pathway and the other probably from phosphatidylcholine; (iii) several isoforms of PKC translocating to nuclei; (iv) increases in activity of the PI-PLC pathway at two points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P3, which may have several functions, including a role in the transfer of mRNA out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and, in particular, 3-phosphorylated inositol lipids, which are now emerging as possible major players in nuclear signaling.

Nuclear lipid signaling.

There is now abundant evidence for the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope, but are actually within the nucleus, presumably not in a bilayer membrane but instead forming proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol and inositol-1,4,5-trisphosphate [Ins(1,4,5)P(3)]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet clear. Generation of diacylglycerol within the nucleus is believed to recruit protein kinase C to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P(3) may mobilize Ca(2+) from the space between the nuclear membranes and thus increase nucleoplasmic Ca(2+). Less well understood are an increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus: (i) two separate routes of synthesis of phosphatidylinositol-4,5-bisphosphate; (ii) two different sources of diacylglycerol, one being from the PI-PLC pathway, and the other probably from phosphatidylcholine; (iii) several different isoforms of PKC translocating to the nuclei; (iv) increases in activity of the PI-PLC pathway at two different points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P(3), which may have several functions, including a role in the transfer of messenger RNA (mRNA) out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and 3-phosphorylated inositol lipids.

Nuclear Lipid Signaling

There is now abundant evidence for the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope, but are actually within the nucleus, presumably not in a bilayer membrane but instead forming proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet clear. Generation of diacylglycerol within the nucleus is believed to recruit protein kinase C to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P3 may mobilize Ca2+ from the space between the nuclear membranes and thus increase nucleoplasmic Ca2+. Less well understood are an increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus: (i) two separate routes of synthesis of phosphatidylinositol-4,5-bisphosphate; (ii) two different sources of diacylglycerol, one being from the PI-PLC pathway, and the other probably from phosphatidylcholine; (iii) several different isoforms of PKC translocating to the nuclei; (iv) increases in activity of the PI-PLC pathway at two different points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P3, which may have several functions, including a role in the transfer of messenger RNA (mRNA) out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and 3-phosphorylated inositol lipids.

Fatal connections: when DNA ends meet on the nuclear matrix.

A damaged nucleus has long been regarded simply as a "bag of broken chromosomes," with the DNA free ends moving around and forming connections with randomly encountered partners. Recent evidence shows this picture to be fundamentally wrong. Chromosomes occupy specific nuclear domains within which only limited movement is possible. In a human diploid nucleus, 6.6 x 10(9) base pairs (bp) of DNA are compartmentalized into chromosomes in a way that allows stringent control of replication, differential gene expression, recombination and repair. Most of the chromatin is further organized into looped domains by the dynamic binding of tethered bases to a network of intranuclear proteins, the so-called nuclear scaffold or matrix. Thus, DNA movement is severely curtailed, which limits the number of sites where interchanges can occur. This intricate organizational arrangement may render the genome vulnerable to processes that interfere with DNA repair. Both lower and higher eukaryotic cells perform homologous recombination (HR) and illegitimate recombination (IR) as part of their survival strategies. The repair processes comprising IR must be understood in the context of DNA structural organization, which is fundamentally different in prokaryotic and eukaryotic genomes. In this paper we first review important cellular processes including recombination, DNA repair, and apoptosis, and describe the central elements involved. Then we review the different DNA targets of recombination, and present recent evidence implicating the nuclear matrix in processes which can induce either repair, translocation, deletion, or apoptosis. J. Cell. Biochem. Suppl. 35:3-22, 2000.

Association with the cellular export receptor CRM 1 mediates function and intracellular localization of Epstein-Barr virus SM protein, a regulator of gene expression.

Splicing and posttranscriptional processing of eukaryotic gene transcripts are linked to their nuclear export and cytoplasmic expression. Unspliced pre-mRNAs and intronless transcripts are thus inherently poorly expressed. Nevertheless, human and animal viruses encode essential genes as single open reading frames or in the intervening sequences of other genes. Many retroviruses have evolved mechanisms to facilitate nuclear export of their unspliced mRNAs. For example, the human immunodeficiency virus RNA-binding protein Rev associates with the soluble cellular export receptor CRM 1 (exportin 1), which mediates nucleocytoplasmic translocation of Rev-HIV RNA complexes through the nuclear pore. The transforming human herpesvirus Epstein-Barr virus (EBV) expresses a nuclear protein, SM, early in its lytic cycle; SM binds RNA and posttranscriptionally activates expression of certain intronless lytic EBV genes. Here we show that both the trans-activation function and cytoplasmic translocation of SM are dependent on association with CRM 1 in vivo. SM is also shown to be associated in vivo with other components of the CRM 1 export pathway, including the small GTPase Ran and the nucleoporin CAN/Nup214. SM is shown to be present in the cytoplasm, nucleoplasm, and nuclear envelope of transfected cells. Mutation of a leucine-rich region (LRR) of SM inhibited CRM 1-mediated cytoplasmic translocation and SM activity, as did leptomycin B, an inhibitor of CRM 1 complex formation. Surprisingly, however, leptomycin B treatment and mutation of the LRR both led to SM becoming more tightly attached to intranuclear structures. These findings suggest a model in which SM is not merely a soluble carrier protein for RNA but rather is bound directly to intranuclear proteins, possibly including the nuclear pore complex.

Transcription Factor Pit-1 and Its Clinical Implications

This review presents a transcription factor, Pit-1, and its relevance to clinical practice. Our knowledge of the molecular and genetic basis of disease is growing at an amazing pace. Intranuclear transcription factor proteins, which influence gene expression, are being discovered and characterized. Pit-1, a pituitary-specific transcription factor, is necessary for the development and subsequent function of somatotrophs, lactotrophs, and thyrotrophs of the anterior pituitary gland. In this review we will discuss the molecular mechanisms by which Pit-1 influences gene expression and explore the role of this factor in pituitary diseases such as hypopituitarism and pituitary tumors.

EFFECT OF ACTINOMYCIN-D ON THE CELL-CYCLE PROGRESSION AND THE EXPRESSION OF P53, WAF1/CIP1, GADD45, AND MDM-2 GENES IN HUMAN ORAL KERATINOCYTES - IMPLICATION OF HUMAN PAPILLOMAVIRUS INFECTION

Exposure of human oral keratinocytes immortalized by transfection with 'high risk' HPV DNA to chemical carcinogens converts the cells to malignant phenotype, but it does not transform normal cells. To investigate the underlying mechanism for different chemical carcinogen susceptibility of normal and the HPV-immortalized oral keratinocytes to genotoxic agent, we studied the progression of cell cycle and the expression of p53, WAF1/CIP1, gadd45 and mdm-2 genes in normal and in the HPV-immortalized oral keratinocytes after exposing cells to actinomycin D. Normal oral keratinocytes demonstrated transient G(1) arrest after the exposure, but the HPV-immortalized cells did not. Actinomycin D significantly increased the levels of intranuclear wild-type p53 and mdm-2 proteins and the transcripts of WAF1/CIP1, gadd45 and mdm-2 in normal cells, but it did not increase them in the HPV-immortalized cells. These data indicate that actinomycin D-induced transient cell cycle arrest may be associated with enhanced level of wild-type p53 protein and the transcripts of WAF1/CIP1 and gadd45 in normal human oral keratinocytes. This study also suggests that overexpressed mdm-2 may not be enough to abrogate the cell cycle arrest mediated by wild-type p53. Our data support the idea that conversion of the HPV-immortalized oral keratinocytes to tumorigenic cells may, in part, be due to the incapability of the cells to arrest the cell cycle when exposed to genotoxic agents, which would result in improper repair of damaged DNA and subsequent inheritance of genetic error.

Nucleolar organizer regions as prognostic indicators in renal cell carcinoma

Nucleolar organizer regions (NORs) are agyrophilic intranuclear proteins associated with sites of rDNA transcription. Several studies have shown NCR numbers to be related to proliferative activity in both benign and malignant neoplasms. Blocks from 182 cases of renal cell carcinoma presenting at Wellington hospital between 1954 and 1983 were retrieved from file. Sections were cut at 30 thickness and stained by the silver-colloid method. NORS were quantified using oil immersion light microscopy. The tumours were staged according to kobson's classification (48 stage I, 26 stage II, 33 stage III and 75 stage IV) and mean NOR numbers for each tumour were correlated with five year survival. Patients with localizec tumours (stage I) and low NCR numbers had 100% survival over five years. Patients with clinical evidence of metastases at presentation (stage IV) showea a high mortality although those with low mean IvCRs had a significantly increaseo survival time. Analysis of data using the log rank test showed NORs to be a significant predictor of survival over the whole series (p=0.0001) and within each of Robson's stages (stage I p=0.0008, stage II p=0.0154, stage III p=0.0009, stage IV p=0.0001). NORs were also found to be incependent of stage as a predictor of survival using Cox's proportional hazard model.

Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products.

A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well-characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as might occur with antigens that are components of subcellular particles.

Sex steroid receptors in lymphoid cells of human endometrium.

Sex steroid hormone action on target tissues is mediated through binding of estrogen and progesterone to specific intranuclear proteins, the estrogen and progesterone receptors (ER and PR). Therefore, in the present report the authors investigated for the presence of ER and PR in lymphoid cells of endometrial stroma that may serve as potential targets for estrogen- and progesterone-mediated effects in endometrium. The presence of ER was shown in nine proliferative and ten secretory endometria and the presence of PR in three secretory and one proliferative endometria. ER and PR were localized by monoclonal antibodies, to the nuclei of cells, with the use of, respectively, peroxidase-antiperoxidase (PAP) and avidin-biotin-peroxidase complex (ABC) methods. Lymphoid cells were then delineated by decoration of their plasma membranes with the use of monoclonal antibodies to HLA-DR, leukocyte common antigen (LCA), Leu-4 (CD3), and IL-2 receptor molecules with the use of an ABC staining procedure. A group of cells in the lymphoid aggregates in endometrial stroma showing membranous staining for HLA-DR, LCA, and Leu-4 molecules had nuclear ER. IL-2 receptor-positive cells were rare in endometrium, and no PR-positive cells were found in lymphoid aggregates. Furthermore, HLA-DR and ER were expressed in the glandular and surface epithelium in the proliferative phase and in occasional glands in the basalis in the late secretory phase. The presence of an ER-positive lymphoid cell population in endometrial lymphoid aggregates suggests that these cells may serve as target cells for estrogen.

Synthesis of the intranuclear crystals in Scolopendrium vulgare (L.) SM

Proteinaceous intranuclear crystals are found in the fern Scolopendrium vulgare. During mitosis these crystals are eliminated from the nucleus into the cytoplasm, where they are dissolved. New crystals appear in the nucleus. The site of synthesis of intranuclear crystal proteins was investigated using quantitative ultrastructural autoradiography after incubation with tritiated lysine. The results suggest a migration of cytoplasmic proteins to the nucleus, part of which would then be incorporated into the intranuclear crystals.

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.

Isolation and characterization of nuclear ghosts from cultured HeLa cells

Macromolecular complexes, which appear as ghosts when viewed by phase contrast microscopy, have been isolated from the nuclei of HeLa cells grown in culture. The preparation of these ghosts involves a detergent wash which removes the unit membranes of the nuclear envelop structure but leaves intact both the nuclear pores and the dense structure conferring nuclear margins (possibly the dense lamella). Detergent-washed nuclei are subsequently treated with 0.5 M MgCl2 and fractionated on continuous sucrose gradients containing 0.5 M MgCl2. The ghosts are recovered as a sharp band at an apparent sucrose density of 47-52% and consist of 72% protein, 10% phospholipid, 14% DNA, And 4% RNA. The release of the majority of intranuclear components is indicated by the large loss of nuclear DNA (95%), RNA (71%), and protein (87%) contrasted to the small loss of phospholipid (27%) druing the conversion of detergent washed nuclei to isolated ghosts. Sodium dodecyl sulfate-polyacrylamide gel patterns of the ghost proteins consist of two major bands with approximate molecular weights of 20,000 and 35,000. The isolation of ghosts with a similar density and protein composition from nondetergent-washed nuclei indicates that the ghost is not an artifact induced by the detergent treatment. The absence of cytoplasmic contamination in the preparations of detergent washed nuclei and nuclear ghosts was demonstrated by chemical, enzymatic, and electron microscope studies. We suggest that the isolated ghosts represent a structural macromolecular complex which underlies and is probably attached to the inner nuclear membrane of intact nuclei. The possible additional presence of intranuclear network proteins has not been excluded.