Nuclear Protein Matrix Research Articles

An immunochemical study of localization of some nuclear matrix proteins in the composition of perinucleolar chromatin

In nuclei of interphase pig embryo kidney cells (PEK), intense fluorescence of fluorochrome DAPI is observed at the periphery of nucleoli in regions of perinucleolar chromatin as a brightly fluorescing rim. In interphase cells, antibodies to proteins with molecular masses of 27, 38, 40, 50, and 65 kDa obtained from sera of patients with autoimmune diseases stain only nuclei as small accumulations or granules. These antibodies do not stain nucleoli themselves, but they are revealed at their periphery as a brightly fluorescing circle in the zone of the perinucleolar chromatin. Antibodies to the nucleolar proteins fibrillarin and B23 specifically stain only nucleoli. In nuclei, after removal of histones and DNA (nuclear protein matrix (NPM)), the protein fibrillarin is distributed uniformly throughout the volume of residual nucleoli, while the protein B23 is revealed only at their periphery where the perinucleolar chromatin is located. Antibodies to proteins with molecular masses of 40, 50, and 65 kDa are connected with the periphery of residual nucleoli and are spread throughout the cell karyoplasm as small accumulations. Antibodies to proteins with molecular masses of 27 and 38 kDa are connected only with the periphery of residual nucleoli. It has been established that the nuclear matrix proteins with molecular masses of 27, 38, 40, 50, and 65 kDa, as well as the protein B23, exist in the composition of the perinucleolar chromatin, which may be a special chromosomal domain associated with nucleolus functioning.

Inflammation and Tooth Movement: The Role of Cytokines, Chemokines, and Growth Factors

When an orthodontic force is applied, the periodontal tissues express extensive macroscopic and microscopic changes, leading to alterations in 5 microenvironments: extracellular matrix, cell membrane, cytoskeleton, nuclear protein matrix, and genome. Capability of adaptive reaction to applied mechanical loading relies in the DNA of periodontal ligament (PDL) and alveolar bone cells. However, an inflammatory process is a precondition for these modifications to occur, which will lead to orthodontic tooth movement (OTM). PDL's vascularity and blood flow changes, as well as mechanical alterations in the cytoskeleton of PDL and bone cells, will result in local synthesis and release of various key mediators, such as chemokines, cytokines, and growth factors. These molecules will induce many cellular responses by various cell types in the periodontium, providing a favorable microenvironment for bone resorption or deposition and, consequently, for OTM. In these inflammation and tissue remodeling sites, cells may also communicate with one another through the interaction of cytokines and other related molecules. The aim of this review is to bring focus on the role of these important local inflammatory mediators that are closely related to the mechanotransduction involved in OTM.

Stabilization of non-histone proteins by copper ions does not alter chromatin properties of polythene chromosomes of Chironomus plumosus

Our previous study showed that the body of polythene chromosomes can be identified even after removal of all histones and DNA in the presence of 2 mM CuCl2; this suggested that copper ions stabilized the bonds between non-histone proteins. In this study we tried to find out if copper ions bind with non-histone proteins reversibly or irreversibly. It is shown that the bodies of normal chromosomes and chromosomes stabilized by 2 mM CuCl2 swell with partial disappearance of the banding pattern in a hypotonic solution (0.055 M NaCl) without copper ions. The selective removal of bivalent cations by 10 mM EDTA solution resulted in decondensation of normal polythene and stabilized chromosomes. The treatment of nuclear protein matrix of polythene chromosomes preparations with 10 mM EDTA resulted in the swelling of polythene chromosome body and disappearance of the banding pattern but their morphological organization maintained.

Nuclear matrix proteins (with molecular masses of 38 and 50 kDa) are transported by chromosomes in mitosis

Using the immunofluorescence method, sera M-68 and K-43 from patients with autoimmune diseases were shown to stain interphase nuclei and the periphery of mitotic chromosomes of pig embryo kidney cells. Western blotting revealed a polypeptide with a molecular mass of 50 kDa in M-68 serum and polypeptide with a molecular mass 38 kDa in K-43 serum. In the nuclear protein matrix, the antibodies to protein with a molecular mass of 38 kDa stained only the nucleolar periphery, while the antibodies to protein with a molecular mass of 50 kDa stained not only the nucleolar periphery, but also all interphase nuclei. It was shown that, among all components of the nuclear protein matrix (lamina, internuclear network, residual nucleoli), only the nucleolar periphery contained the 38-kDa protein, while the 50-kDa protein was part of the residual nucleolar periphery and participated in the formation of a nuclear-protein network. Both proteins in interphase cell in situ were located in nuclei, but one of them with a molecular mass of 50 kDa was in the form of small, clearly outlined granules, while the other protein (38 kDa) was in the form of small, bright granules on a background of a diffusely stained nucleus. Both proteins also were revealed as a continuous rim around the nucleolar periphery. During all mitotic stages, the 50-kDa protein was seen over the whole chromosomal periphery as a sheath, while the 38-kDa protein formed individual fragments and granules around them. After the decondensation of the nucleus and chromosomes induced by hypotonic treatment, both antibodies stained interphase nuclei diffusely, whereas, in mitotic cells, they stained the surfaces of swollen chromosomes. Polypeptide with a molecular mass of 50 kDa maintained a strong connection with the periphery of the chromosome in the norm during decondensation induced by hypotonic treatment and during subsequent recondensation in isotonic medium, while, during recondensation, protein with a molecular mass of 38 kDa partially lost contact with the chromosome and, at the same time, appeared in the form of granules in the cytoplasm. The obtained data allow one to conclude that nuclear matrix proteins can be transferred with peripheral chromosomal material; similar to the main nucleolar proteins (fibrillarin, B-23, nucleolin, et al.) and some non-nucleolar components of the nuclear protein matrix, they can also have connections of different stabilities with chromosomal periphery.

Nuclear protein matrix of giant nuclei of polythene chromosome organization of midge Chironomus plumosus determinates

Giant nuclei from salivary glands of the midge Chironomus plumosus were treated in situ with 2M NaCl detergent and nucleases to reveal residual nuclear matrix proteins (NMP). It was shown that, after the prestabilization of nonhistone proteins with 2 mM CuCl2, the polythene chromosome body preserved its morphologic integrity and banding pattern, even after the extraction of all histones and DNA. The stabilized NPM of polythene chromosomes can be observed in both light and electron microscopy; no interchromatin fibrillary-granular structures are revealed in the nucleus except for peripheral lamina. Using the immunocytochemical method, in polythene chromosomes, we managed to detect major nonhistone proteins (topoisomerase IIα and SMC 1) and some RNA-components. Besides, in giant nuclei of larvae of early stages there is observed BrDU incorporation visualizing sites of DNA synthesis, which also are connected with NPM of polythene chromosomes. Thus, it can be concluded that structure of NPM of giant nuclei of Chironomus plumosus has all properties of NPMs of usual interphase nuclei; furthermore, this NPM determines specific structure of the polythene chromosome.

Nuclear protein matrix in giant nuclei from salivary glands of Chironomus plumosus

Polythene chromosomes from salivary glands of Chironomus plumosus were treated in situ in order to reveal residual nuclear protein matrix (NPM). It was shown that after the removal of H1-histones by 0.6 M NaCl the general morphology of chromosomes is preserved, revealing distinct banding pattern. Further treatment of chromosomes with 2 M NaCl and DNase completely disorganized the structure of chromosome bodies and patterns of banding. Preliminary treatment of salivary glands with 2 mM CuCl2 resulted in stabilization of the structure of polythene chromosome in every stage of histone and DNA extractions. Stabilized chromosomes maintained their morphology and banding patterns observed by phase contrast or after the staining with Brilliant blue. Thus, after the removal of histones and DNA, stabilized chromosomes retain their morphological features, which depend on the presence of NMP. In stabilized polythene chromosomes, in spite of the absence of histones and DNA, topoisomerase IIα retains its localization, typical for untreated chromosomes.

Simultaneous Observation of DNA Fragmentation and Protein Loss in the Boar Spermatozoon Following Application of the Sperm Chromatin Dispersion (SCD) Test

DNA fragmentation and the nuclear protein matrix in boar spermatozoa were simultaneously assessed using a specific variant of the sperm chromatin dispersion (SCD) test that allows direct visualization of DNA and nuclear proteins under standard conditions of chemical lysis. Nuclear proteins remaining after lysis were stained with the fluorochrome 2,7-dibrom-4-hydroxy-mercury-fluorescein for specific protein staining. DNA and nuclear protein were stained in control-untreated (no lysis) and treated sperm cells (lysis), resulting in the identification of 3 cell types: type 1: nonlysed (control-untreated) cells; type 2: lysed cells showing nonfragmented DNA; and type 3: lysed cells showing fragmented DNA. DNA damage was also purposely induced by incubating the sperm in 0.015% H(2)O(2) for 48 hours at 37 degrees C; the cells were correspondingly stained for DNA fragmentation and protein. Nonlysed control sperm (type 1) nuclei showed no halos and stained strongly for protein in the postacrosomal region. Lysed spermatozoa with nonfragmented DNA (type 2) showed evidence of restricted DNA loop dispersions at the caudal extremity of the sperm head and a more homogenous but similar distribution of protein matrix in comparison with untreated spermatozoa. Lysed spermatozoa with fragmented DNA (type 3) exhibited large halos of DNA loops and a loss of the nuclear protein matrix component. Sperm cells exposed to 48 hours' incubation at 37 degrees C and then treated with the lysing agent showed a concurrent and progressive loss of nuclear protein in association with correspondingly increased levels of DNA fragmentation. Discriminant analysis of quantitative fluorescence using digital image analysis and conducted after SCD processing revealed that DNA fragmentation and protein could be evaluated in an automated system. Ninety-seven percent of the total analyzed cells were accurately classified according to previously defined cell types (1, 2, and 3). The results of the current study demonstrated a synergistic relationship between that of nuclear protein alteration and DNA damage in the boar sperm cell. The importance of abnormal nuclear protein alteration to DNA fragmentation and any related effect on fertility remains to be investigated.

Evidence of modified nuclear protein matrix in human spermatozoa with fragmented deoxyribonucleic acid

Human spermatozoa were processed for determination of DNA fragmentation with use of an in situ diffusion assay, so that those cells containing DNA fragmentation produce extensive peripheral dissemination of DNA fragments after lysis in an agarose microgel. Quantification of specific protein staining confirmed that sperm cells without DNA fragmentation had almost complete removal of nuclear matrix proteins, whereas spermatozoa with DNA fragmentation tended to retain residual nucleoskeletal protein in a collapsed and condensed state. This result suggests that a modified nuclear protein matrix associates with fragmented sperm DNA.

Current concepts in the biology of orthodontic tooth movement

Adaptive biochemical response to applied orthodontic force is a highly sophisticated process. Many layers of networked reactions occur in and around periodontal ligament and alveolar bone cells that change mechanical force into molecular events (signal transduction) and orthodontic tooth movement (OTM). Osteoblasts and osteoclasts are sensitive environment-to-genome-to-environment communicators, capable of restoring system homeostasis disturbed by orthodontic mechanics. Five micro-environments are altered by orthodontic force: extracellular matrix, cell membrane, cytoskeleton, nuclear protein matrix, and genome. Gene activation (or suppression) is the point at which input becomes output, and further changes occur in all 5 environments. Hundreds of genes and thousands of proteins participate in OTM. Gene-directed protein synthesis, modification, and integration form the essence of all life processes, including OTM. Bone adaptation to orthodontic force depends on normal osteoblast and osteoclast genes that correctly express needed proteins at the right times and places. Cell membrane receptor-ligand docking is an important initiator of signal transduction and a discovery target for new bone-enhancing drugs. Despite progress in identification of regulatory molecules, the genetic mechanism of "orchestrated synthesis" between different cells, tissues, and systems remains largely unknown. Interpatient variation in mechanobiological response is most likely due to differences in periodontal ligament and bone cell populations, genomes, and protein expression patterns. Discovery of mutations in OTM-associated genes of orthodontic patients, including those regulating osteoclast bone-matrix acidification, chloride channel function, and osteoblast-derived mineral and protein matrices, will permit gene therapy to restore normal matrix and protein synthesis and function. Achieving selectivity in targeting abnormal genes, cells, and tissues is a major obstacle to safe and effective clinical application of gene engineering and stem-cell mediated tissue growth. Orthodontic treatment is likely to evolve into a combination of mechanics and molecular-genetic-cellular interventions: a change from shotgun to tightly focused communication with OTM cells.

Comparative Evaluation of the Nuclear Matrix Protein, Fibronectin, Urinary Bladder Cancer Antigen and Voided Urine Cytology in the Detection of Bladder Tumors

We evaluate the diagnostic efficacy of nuclear matrix protein-22 (NMP22, Matritech, Newton, Massachusetts), fibronectin and urinary bladder cancer antigen (UBC, IDL Biotech, Borlange, Sweden) compared with voided urine cytology in the detection of bladder cancer. A total of 168 patients provided a single voided urine sample for NMP22, fibronectin an ideal monoclonal for urinary bladder cancer and cytology before cystoscopy. Cystoscopy was done for all patients as the reference standard for identification of bladder cancer. Biopsy of any suspicious lesion was performed for histopathological examination. Of the 168 cases 100 were histologically diagnosed as bladder cancer, whereas the remaining 68 had benign urological disorders. A group of 47 healthy volunteers were also enrolled in this study. Voided urine was evaluated by NMP22, fibronectin and UBC, and their values were expressed relative to mg. creatinine. The optimal threshold values for NMP22, fibronectin and UBC were calculated by receiver operator characteristics curves as 27 units per mg. creatinine, 198 mg./mg. creatinine and 13 ng./mg. creatinine, respectively. The levels and positive rates of the 3 parameters were significantly higher in the malignant group compared to either the benign group or normal controls. Of the entire group NMP22, fibronectin and UBC were positive in 93.2%, 91% and 68.2%, respectively in bladder cancer cases with positive cytology. Moreover, these positive rates were significantly higher in bilharzial bladder cancer cases (58.8%, 67.5%, 58.8%, respectively) compared to nonbilharzial cases (35.6%, 36.3%, 31.1%). Overall sensitivity and specificity were 85% and 91.3% for NMP22, 83% and 82.6% for fibronectin, 67% and 80.8% for UBC and 44% and 100% for voided urine cytology. Combined sensitivity of voided urine cytology with the 3 biomarkers together was higher than either combined sensitivity of voided urine cytology with 1 of the biomarkers or than that of the biomarker alone. Our data indicate that NMP22 and fibronectin had superior sensitivities compared to UBC and voided urine cytology, while NMP22 and voided urine cytology had the highest specificities. The combined use of markers increased the sensitivity of cytology from 44% to 95.3%. The higher sensitivities of markers in bilharzial than nonbilharzial bladder cancer highlight their clinical use in screening patients with urinary bilharziasis.

IN VITRO AND IN VIVO EFFECTS OF VITAMIN D (CALCITRIOL) ADMINISTRATION ON THE NORMAL NEONATAL AND PREPUBERTAL PROSTATE

Although many studies have investigated the role of calcitriol in the growth regulation of normal and cancerous prostates, little is known about its role in early prostatic development. The interactions between calcitriol and androgens, and their actions on the normal prostate have similarly been proposed but not evaluated. Previous studies in our laboratory have revealed that in utero administration of 1,25-dihydroxycholecalciferol or calcitriol can influence prostate growth and differentiation throughout the life of the animal. We further examined the influence of calcitriol on the normal prostate in vitro and in vivo by focusing on early stages of prostatic development. The effects of calcitriol on the growth of the normal human neonatal prostatic epithelial cell line 267B-1 was determined in the presence and absence of dihydrotestosterone (DHT). We also examined the effect of calcitriol on the growth of maturing rat prostates in vivo. Before puberty 4 groups of rats 27 to 38 days old were treated with vehicle (controls) or calcitriol. When the rats reached adulthood at age 100 to 110 days a control group and a calcitriol group were sacrificed. The other 2 groups were given exogenous DHT for 5 days. For the animals to become adapted to DHT they were kept alive for 1 additional week and sacrificed at about age 120 days. In vitro studies demonstrated that 267B-1 cells possess vitamin D receptors and their growth was inhibited by calcitriol with an IC50 (concentration resulting in 50% cytotoxicity) of 30 microM. Proliferation of these neonatal prostate cells was also inhibited by calcitriol in the presence of DHT in vitro. Our studies indicate that, although calcitriol was administered at the apparently important prepubertal period, there was no difference in prostatic weights between the control and calcitriol treated rats. Exogenous administration of DHT decreased prostatic weight of control rats but in rats treated with 1,25-dihydroxycholecalciferol DHT did not have any significant effect on prostatic weight. No statistically significant differences were observed in seminal vesicle weights among the different groups of animals. Analysis of the nuclear matrix protein composition of the prostatic tissue showed differences in composition between the DHT, and calcitriol and DHT treated rat prostates. These studies indicate that calcitriol administered just before puberty does not significantly influence prostatic growth in the presence of endogenous or exogenous administered DHT, and has an inhibitory effect on neonatal prostate epithelial cell growth in vitro in the presence and absence of DHT. Treatment with calcitriol and DHT also results in differences in nuclear matrix protein composition. Prepubertal administration of calcitriol may inhibit the exogenous DHT action in decreasing epithelial growth and stimulating stromal proliferation in the rat prostate.

Regions of the Haptoglobin 5' Flanking Gene Sequence Show Different Binding Affinities to Nuclear Matrix Proteins during the Acute Phase Response

The effect of the acute phase response on the affinity of binding between nuclear matrix proteins and the rat haptoglobin (Hp) gene region was examined. Nuclear matrices isolated from acute phase livers were enriched with the 5' Hp gene flanking region (-705/+159), but not with the spliced, protein-coding sequence. Reassociation experiments with isolated nuclear protein matrix spheres and end-labeled fragments I (-146/+156), II (-146/-541), and III (-541/-705) revealed that the matrix proteins displayed an increased binding potential during the acute phase response for all of the examined regions, this being most pronounced for fragment II. BAL 31 digestion of fragment II showed that the sequence element that was responsible for the increased association with nuclear matrix proteins during the acute phase response was a tract of 38 adenine bases. The DNA region established stable associations with nuclear lamin B (67 kDa, pI 5.7) in the controls, and with lamins A (69 kDa, pI 7.0), B, isoforms of lamin C (62 kDa, pI 6.55-6.95), and a 55-kDa (pI 5.9) polypeptide during the acute phase response. Sequence ABC (-165/-56), which overlaps fragments I and II and represents the Hp cis-acting element, did not bind to the non-histone nuclear matrix proteins.

Neutral Filter Elution Detects Differences in Chromatin Organization Which Can Influence Cellular Radiosensitivity

We have shown previously that the neutral filter elution assay is dependent not only on the number of DNA double-strand breaks present in a mammalian cell but also on the way in which DNA expands on the filter following lysis. Results in this study indicate that the rate of DNA elution appears to be dependent upon the proximity of the DNA in relation to the replication complex. The rate of elution for DNA analyzed immediately after a 30-min labeling period with [14C]thymidine was about five times slower than the rate of elution for bulk-labeled DNA. However, the rate was increased a few hours later when the recently replicated DNA had matured and was likely to be farther from replication-associated attachment sites on the nuclear protein matrix. About one cell cycle after pulse labeling, when the labeled DNA was replicated again, DNA underwent similar changes in elution rate. For the four cell lines examined here, the elution rate 3-4 h after pulse labeling correlated with cellular radiosensitivity. Changes in rate of elution caused by altering EDTA concentration or pH may also be explained by DNA structural changes which occur during lysis. We conclude that the neutral filter elution method is sensitive to differences in chromatin organization which may also play a role in cell sensitivity to ionizing radiation.

Distribution and metabolism of estramustine in HeLa cells and the human prostatic tumour cell line 1013L

The metabolism of estramustine [estradiol-3- N-bis(2-chloroethyl) carbamate] was investigated in the human prostatic tumour cell line 1013L and the human cervix tumour cell line HeLa S3. Uptake studies revealed that estramustine (EM), and its 17-ketoanalogue estromustine (EoM), differed in their nuclear binding pattern in 1013L cells but not in HeLa cells. Most of the nuclear radioactivity from both EM and EoM was found in the fraction containing the majority of the phospholipids. HPLC studies on EM-treated 1013L cells showed the presence of the oxidized metabolite EoM, in the medium, an enrichment of estradiol and estrone in whole cells and EM and EoM bound to the nuclear protein matrix. Similar studies on the HeLa cell line showed a completely different pattern, no metabolites other than EoM were found in the cell medium and whole cells but several very lipophilic metabolites were found bound to the nuclear protein matrix. On investigation of other tumour cell lines these metabolites were found to be unique to HeLa cells. The results extend our knowledge concerning EM and demonstrate that the cell line 1013L is a relevant model system for studying drugs active against human prostatic tumours.

Identification of nuclear envelope proteins and glycoproteins which co-isolate with the nuclear protein matrix

Nuclear envelopes and nuclear matrices were isolated from rat liver nuclei. Although differences in polypeptide composition of the structures are evident on SDS gel electrophoresis, they have an almost identical distribution of concanavalin A-binding glycoproteins. These matrix-associated concanavalin A-binding glycoproteins derive entirely from the nuclear envelope and are recovered almost quantitatively in the matrix. They constitute easily identifiable markers for nuclear envelope association with matrix or other nuclear subfractions. Surface labelling of nuclei with 125I using solid-phase lactoperoxidase further confirmed that a large number of envelope-associated nuclear surface proteins co-isolate with the matrix. Protein kinase activity, as well as endogenous substrates for the kinase(s) are shown to be the same in both envelopes and matrix. Envelope-derived proteins and glycoproteins may comprise a substantial proportion of total matrix protein.

Nuclear protein matrix as a target for estramustine-induced cell death.

The effect of estramustine [estradiol 3-N-bis(2-chloroethyl)carbamate] on the human prostatic tumor cell line 1013L was investigated. Cell proliferation experiments revealed that estramustine cytotoxicity varied during the different phases of cell growth. Maximum cell killing was found in early log phase, but cell death also occurred in the stationary phase. Mitotic arrest was found at cytotoxic concentrations throughout the log phase. Subcellular distribution studies showed that the cellular uptake of estramustine increased throughout the log phase and remained steady during the stationary phase. Nuclear uptake in contrast was similar in all phases, whereas a preferential binding to the nuclear protein matrix was found to increase throughout the log phase and even during the stationary phase of growth. This implicates the nuclear protein matrix as a target for estramustine cytotoxicity.

Blood Cell Nuclei: The Structure and Function of Lymphoid and Erythroid Nuclei

Publisher Summary This chapter describes the functional organization of the nucleus and the nuclear protein matrix of lymphocyte and erythrocyte. It also presents an ultrastructure of human bone marrow cell maturation and a survey of the genome activity and gene expression in avian erythroid cells. Despite the inherent differences in nucleic acid metabolism in the developing erythroid and lymphoid lineages and in their mature cells, certain common features are also observed with respect to the structure and function of the nuclear envelope, nucleoli, nuclear matrix, and chromatin. The contribution of electron microscopic studies to the understanding of lymphoid and erythroid nuclear structure is well recognized. Studies on the mammalian and avian lymphocyte clearly indicate that for both the T and B cell lineages, fully differentiated nondividing mature cells are ultimately produced. These cells respond to an apparently infinite variety of antigenic stimuli to become proliferating cells, which participate in the immunological response. The erythroid lineage, however, yields a nondividing terminally differentiated cell, which, in the case of the mammal, expels its metabolically inactive nucleus within the sinusoids of the bone marrow hematopoietic sites and is released as the circulatory reticulocyte, ultimately becoming the fully mature erythrocyte. During the course of erythroid and lymphoid development, the proportion of dividing cells progressively decreases in parallel with marked changes in mRNA production, chromatin condensation, and overall nuclear volume.

Cell lethality after selective irradiation of the DNA replication fork.

It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125I-iododeoxyuridine (125IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 micrograms/ml) reduced cellular 125IUdR incorporation to 3-5% of the control value. The residual 125I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125I decays at the replication fork to that of 125I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D0: 101 125I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome.

The isolation of nuclear envelope from peripheral lymphocytes: An ultrastructural study

A procedure is presented for the isolation of nuclear envelope from circulatory human lymphocytes. The chromatin of purified lymphocyte nuclei is decondensed by washing with 1 mM sodium bicarbonate buffer (pH 7.2) in the absence of chromatin-stabilizing divalent cations. Following a limited digestion with pancreatic DNAase-I, the nuclear envelope is centrifugally harvested and repeatedly washed in bicarbonate buffer to remove solubilized chromatin. Further purification of the nuclear envelope is achieved by isopycnic banding on sucrose step gradients. The purity and morphology of the isolated nuclear envelope is assessed by negative-staining electron microscopy. All the membranous material present in the purified preparations is found to possess nuclear pore complexes, which are accepted as being morphological ‘markers’ for the nuclear envelope. Relatively intact lympocyte nuclear envelope ‘ghost’ are found to possess a low frequency of nuclear pore complexes together with the presence of nuclear pore complex clustering, in agreement with the literature on freeze-fractured lymphocyte nuclear envelope. Negatively stained fragments of nuclear envelope often contain the nuclear pore complex clusters, indicating that lesions through the outer and inner nuclear membranes have more readily occurred in the regions of envelope devoid of nuclear pores complexes. This observation supports the hypothesis that the nuclear pore complexes act as linking positions between the two nuclear membranes and thereby act as zones of stabilization for the overall nuclear envelope during the isolation of the nuclei and envelope, even though their in vivo function is more likely to be associated with ribonucleoprotein translocation. Electrophoresis of sodium dodecyl sulphate-solubilized lymphocyte nuclear envelope on polyacrylamide gradient gels has revealed the molecular weight range of the principal polypeptides present. Excellent reproducibility of polypeptide content has been achieved for consecutive preparations of lymphocyte nuclear envelope. A comparison of polypeptide content of rat liver and human lymphocyte nuclear envelope is presented. The methodology employed and the data presented is discussed in relation to the available literature on lymphocyte nuclei and nuclear envelope, and also to the existence of a nuclear protein matrix in the transcriptionally inactive nucleus of the lymphocyte.

Nuclear protein matrix of seminal vesicle epithelium

Nuclear protein matrix was isolated from guinea pig seminal vesicle epithelium and liver. The two matrices were similar in fine structure as seen by transmission electron microscopy, in protein electropherograms, and in percent composition relative to protein, DNA, and RNA. Scanning electron microscopy was used to examine intact seminal vesicle nuclei, nuclei after treatment with Triton X-100 and DNAse I, and purified nuclear matrix. The matrix surface presented a ‘porous’ appearance by both scanning and transmission electron microscopy. The matrices of liver and seminal vesicle epithelium (SVE) and the intact nuclei of SVE were assayed for specific binding of free synthetic androgen, 17α-methyltrienolone (R1881). Saturable specific binding was demonstrable for seminal vesicle matrix but not for liver matrix. Maximal binding of androgen occurred at a concentration of approximately 12 nM and was demonstrated to be 1.34 ± 0.22 pmol of R1881 per mg of seminal vesicle matrix protein; the K d was approximately 8nM. The binding of labeled R1881 to matrix could be inhibited with low concentrations of unlabeled androgens, but not with estrogens or other steroids. Our data indicate that the binding of androgen to matrix could account for at least 21% of the binding to intact nuclei.