Non-nuclear Membranes Research Articles

Characterization of muscle ankyrin repeat proteins in human skeletal muscle.

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule.

Atherosclerosis and Vascular Biologic Responses to Estrogens: Histologic, Immunohistochemical, Biochemical, and Molecular Methods.

Atherogenesis is a multifactorial pathologic process influenced by genetics and environmental factors such as diet, exercise, stress, and other exposures. Estrogen receptors (ER) are expressed in cells of the arterial wall, suggesting that estrogen receptor ligands (estradiol, natural and pharmacologic ligands) may directly affect arterial biology and atherogenesis. Ligand bound estrogen receptor alpha and beta (ERα, ERβ) can influence physiology through direct binding to estrogen response elements in the DNA, through interactions with other transcription factors such as NF-κB, or through rapid effects not dependent on gene expression changes but instead through non-nuclear membrane sites involving ERα, ERβ, or G-coupled protein ER (GPER1). Elucidation of potential direct effects of estrogens on the artery wall requires careful evaluation of arterial biologic responses to estrogens. We have developed a comprehensive approach to understand the mechanisms of estrogen action which employs histologic measures of the size and other characteristics of atherosclerotic lesions, immunohistochemical assessments of cellular composition, evaluation of chemical, molecular, and genomic changes in the arterial environment, and determination of the relationships between arterial estrogen receptor expression and atherogenesis. This approach can provide important insights into the mechanisms of action of estrogen and other mediators of atherogenesis.

Characterization of cellular DGK-θ

Characterization of cellular DGK-θ

Distribution of calcium-independent phospholipase A2 (iPLA2) in monkey brain

The present study was carried out to elucidate the distribution of calcium-independent phospholipase A(2) (iPLA(2)) in the normal monkey brain. iPLA(2) immunoreactivity was observed in structures derived from the telencephalon, including the cerebral neocortex, amygdala, hippocampus, caudate nucleus, putamen, and nucleus accumbens, whereas structures derived from the diencephalon, including the thalamus, hypothalamus and globus pallidus were lightly labeled. The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled. Immunoreactivity was observed on the nuclear envelope of neurons, and dendrites and axon terminals at electron microscopy. Western blot analysis showed higher levels of iPLA(2) protein in the cytosolic, than the nuclear fraction, but little or no protein in the membrane fraction. Similarly, subcellular fractionation studies of iPLA(2) activity in rat brain cortical cell cultures showed greater enzymatic activity in the cytosolic, than the nuclear fraction, and the least activity in non-nuclear membranes. The association of iPLA(2) with the nuclear envelope suggests a role of the enzyme in nuclear signaling, such as during neuronal proliferation and differentiation or death. In addition, the localization of iPLA(2) in dendrites and axon terminals suggests a role of the enzyme in neuronal signaling.

Overexpression of PKCε sensitizes LNCaP human prostate cancer cells to induction of apoptosis by bryostatin 1

Phorbol 12-myristate 13-acetate (PMA)-induced apoptosis of androgen sensitive LNCaP human prostate cancer cells is a well known phenomenon that involves prolonged translocation of multiple protein kinase C (PKC) isozymes to nonnuclear membranes. We have shown recently that PMA-induced death of C4-2 cells, androgen hypersensitive derivatives of LNCaP cells, requires both PKCdelta and a redundant pathway that includes PKCs alpha and epsilon. In contrast, it has been reported that overexpression of murine PKCepsilon in LNCaP cells renders those cells resistant to PMA-induced death, as well as androgen insensitive. Here we report that inducible or constitutive overexpression of human PKCepsilon does not alter the sensitivity of LNCaP cells to either PMA or androgen, nor does it alter expression of caveolin-1 or phosphorylated Rb, reported effects of overexpression of murine PKCepsilon. Moreover, overexpression of very high amounts of PKCepsilon sensitized LNCaP cells to induction of apoptosis by bryostatin 1, a non tumor-promoting activator and down-regulator of PKC isozymes that blocks PMA-induced apoptosis of parental LNCaP cells, mimicked our previous results with overexpression of PKCalpha in LNCaP cells. Given reports that overexpression of PKCepsilon is frequent in human prostate tumors, our results may have important implications for a potential prostate cancer therapy.

Phorbol Ester-induced Apoptosis of C4-2 Cells Requires Both a Unique and a Redundant Protein Kinase C Signaling Pathway

Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.

Effect of retinoic acid on the Ca2+-independent phospholipase A2 in nuclei of LA-N-1 neuroblastoma cells.

LA-N-1 neuroblastoma cell cultures contain Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca2+-independent phospholipases A2 hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.

Natural Splicing of Exon 2 of Human Interleukin-15 Receptor α-Chain mRNA Results in a Shortened Form with a Distinct Pattern of Expression

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.

Expression of CCKB/gastrin receptor isoforms in gastro-intestinal tumour cells.

Anti-serum raised against the human cholecystokinin B (CCKB)/gastrin receptor was used in Western blotting to differentiate the cellular locations of receptor isoforms expressed by human gastro-intestinal (GI) tumour cell lines. Using anti-serum directed against the amino-terminal extracellular tail of the CCKB/gastrin receptor, 8/9 cell lines were shown to express immunoreactive proteins of either m.w. 70 or 40 kDa, or both. Both isoforms were found to be associated with intracellular, non-nuclear membranes, whereas only the 70 kDa protein was expressed in the plasma membrane. Receptor expression was related to gastrin production and secretion. Both progastrin and glycine-extended gastrin-17 were produced and secreted by the tumour cell lines; however, carboxy amidated gastrin was not detected by radioimmunoassay. A CCKB/gastrin receptor transfectant NIH3T3 cell line did not produce detectable gastrin and showed exclusive expression of the 70 kDa receptor on the plasma membrane. One cell line had <50 pg/ml cell-associated progastrin and no detectable receptor form. Cell lines expressing 50-150 pg/ml had both 40 and 70 kDa receptor forms. Those expressing >150 pg/ml progastrin had only the 40 kDa isoform, which was shown to be exclusively expressed on intracellular, non-nuclear membranes, in one of the cell lines. Of the 4 cell lines exclusively expressing the lower m.w. receptor, 3 had gastrin present within the cell, which was not secreted. Thus, if cell-associated gastrin induces a proliferative effect, it may be by an intracrine pathway. Our study has identified the presence of CCKB/gastrin receptor isoforms in different cellular locations and may help toward understanding the complex autocrine and intracrine pathways mediated by gastrin peptides.

Expression of CCKB/gastrin receptor isoforms in gastro‐intestinal tumour cells

Anti-serum raised against the human cholecystokinin B (CCKB)/gastrin receptor was used in Western blotting to differentiate the cellular locations of receptor isoforms expressed by human gastro-intestinal (GI) tumour cell lines. Using anti-serum directed against the amino-terminal extracellular tail of the CCKB/gastrin receptor, 8/9 cell lines were shown to express immunoreactive proteins of either m.w. 70 or 40 kDa, or both. Both isoforms were found to be associated with intracellular, non-nuclear membranes, whereas only the 70 kDa protein was expressed in the plasma membrane. Receptor expression was related to gastrin production and secretion. Both progastrin and glycine-extended gastrin-17 were produced and secreted by the tumour cell lines; however, carboxy amidated gastrin was not detected by radioimmunoassay. A CCKB/gastrin receptor transfectant NIH3T3 cell line did not produce detectable gastrin and showed exclusive expression of the 70 kDa receptor on the plasma membrane. One cell line had <50 pg/ml cell-associated progastrin and no detectable receptor form. Cell lines expressing 50–150 pg/ml had both 40 and 70 kDa receptor forms. Those expressing >150 pg/ml progastrin had only the 40 kDa isoform, which was shown to be exclusively expressed on intracellular, non-nuclear membranes, in one of the cell lines. Of the 4 cell lines exclusively expressing the lower m.w. receptor, 3 had gastrin present within the cell, which was not secreted. Thus, if cell- associated gastrin induces a proliferative effect, it may be by an intracrine pathway. Our study has identified the presence of CCKB/gastrin receptor isoforms in different cellular locations and may help toward understanding the complex autocrine and intracrine pathways mediated by gastrin peptides. Int. J. Cancer 77:572–577, 1998. © 1998 Wiley-Liss, Inc.

The distribution and metabolism of arachidonate-containing phospholipids in cellular nuclei.

The cell nucleus has been identified as a location to which several arachidonic acid-metabolizing enzymes are located in stimulated cells. However, little information exists describing the distribution of arachidonate-containing phospholipids associated with the nucleus or the control of their composition. In this study, nuclei isolated from human monocyte-like THP-1 cells were found to have a distribution of arachidonyl-phospholipids which is markedly different from that of other cellular membranes. THP-1 nuclei which contained 22% of total cellular arachidonate, showed a near equal distribution of arachidonate in 1-acyl-2-arachidonoyl-glycero-3-phosphocholine, 1-acyl-2-arachidonyl-glycero-3-phosphoethanolamine, 1-acyl-2-arachidonoyl-glycero-3-phosphoinositol and 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species. In contrast in non-nuclear membranes, arachidonate was located primarily in 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species which accounted for approximately half of the arachidonate in all non-nuclear phospholipids. Isolated nuclei were incapable of initially acylating arachidonic acid into their phospholipids in the absence of cellular cytosol. However, they were capable of efficiently remodelling existing arachidonate between phospholipid classes and subclasses. Isolated nuclei contained 25-30% of the cellular activity of CoA-independent transacylase, the key enzyme responsible for arachidonate-phospholipid remodelling. This enzyme is also critical in the control of arachidonate availability following cell stimulation. Given that the cellular distribution of arachidonate is such that nuclei are enriched in donor substrates for the CoA-independent transacylase reaction, that non-nuclear membranes are enriched in acceptor substrates and that nuclei have the enzymatic machinery to remodel arachidonate efficiently, these results suggest that CoA-independent transacylase may be responsible for the remodelling of arachidonate not only between different phospholipid species within the same organelles but also between different sub-cellular compartments.

HL60 nuclei lacking the nuclear double membrane contain a PLD activity which is insensitive to the ADP-ribosylation factor.

Conference Article| November 01 1997 46 HL60 nuclei lacking the nuclear double membrane contain a PLD activity which is insensitive to the ADP-ribosylation factor JOANNA M. CLARK; JOANNA M. CLARK 1CRC Institute for Cancer Studies, Clinical Research Block, University of Birmingham, Birmingham B15 2TA, U.K. Search for other works by this author on: This Site PubMed Google Scholar MATTHEW N. HODGKIN; MATTHEW N. HODGKIN 1CRC Institute for Cancer Studies, Clinical Research Block, University of Birmingham, Birmingham B15 2TA, U.K. Search for other works by this author on: This Site PubMed Google Scholar MICHAEL J.O. WAKELAM MICHAEL J.O. WAKELAM 1CRC Institute for Cancer Studies, Clinical Research Block, University of Birmingham, Birmingham B15 2TA, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (4): S590. https://doi.org/10.1042/bst025s590 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation JOANNA M. CLARK, MATTHEW N. HODGKIN, MICHAEL J.O. WAKELAM; 46 HL60 nuclei lacking the nuclear double membrane contain a PLD activity which is insensitive to the ADP-ribosylation factor. Biochem Soc Trans 1 November 1997; 25 (4): S590. doi: https://doi.org/10.1042/bst025s590 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: PLD, phospholipase D, rARF1, recombinant, myristoylated ADP-ribosylation factor 1, ER, endoplasmic reticulum, Grp, glucose-regulated protein, PC, phosphatidylcholine This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis.

Cytosolic phospholipase A2 (cPLA2) is a good candidate for mediating the agonist-stimulated release of arachidonic acid (AA) from membrane phospholipids. This enzyme undergoes a Ca(2+)-dependent translocation from the cytosol to a membrane site in a variety of cell types, and this site has recently been identified as the nuclear envelope in leucocytes. The functional correlate of this finding has not yet been established. The present study was therefore undertaken to determine whether translocation of cPLA2 to the nuclear envelope was associated with localized phospholipid hydrolysis at this site. Rat alveolar epithelial cells, previously shown to contain cPLA2, were prelabelled with [3H]AA and stimulated with the model agonist, ionophore A23187. Ionophore-induced AA release exhibited characteristics typical of a cPLA2-mediated response, in that it was Ca(2+)-dependent, sn-2 AA-selective, and inhibited by arachidonyl trifluoromethyl ketone. As determined by indirect immunofluorescence microscopic analysis as well as subcellular fractionation with immunoblotting, ionophore treatment resulted in a translocation of cPLA2 protein from the cytoplasm to the nuclear envelope. To determine whether the nuclear membrane was indeed the source of released AA, prelabelled cells were incubated in the presence or absence of A23187, after which the phospholipid radioactivity was quantified in nuclear and non-nuclear membrane fractions. [3H]AA was distributed in both nuclear and non-nuclear membrane phospholipids. Following A23187 stimulation, the loss of [3H]AA from nuclear membrane phospholipids accounted for 88.1 +/- 5.8% of the total loss from phospholipids and for 92.9 +/- 2.3% of the total [3H]AA released into the medium. These results demonstrate for the first time that agonist-stimulated translocation of cPLA2 to the nuclear envelope is associated with phospholipid hydrolysis which is preferentially localized to that site.

Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells.

Arachidonate metabolism by 5-lipoxygenase (5-LO) coincides with the translocation of the enzyme from a soluble to a pelletable fraction in thoroughly disrupted granulocytic cells. While immunoelectron microscopy has identified the nuclear membrane as the site at which 5-LO, as well as 5-LO activating protein (FLAP), are localized in activated cells, the locale of soluble 5-LO in unstimulated cells could not be established by this technique. We asked whether the nucleus might also be the site for soluble 5-LO in unstimulated cells, and utilized rat basophilic leukemia (RBL) cells as model granulocytic cells to address this question. Using three different techniques to disrupt cells while leaving nuclei intact (mild nitrogen cavitation, Dounce homogenization, and detergent lysis), immunoblot analysis indicated abundant 5-LO in isolated nuclei. Within purified nuclei, 5-LO existed in two pools: a soluble pool that was readily released upon nuclear disruption and a bound pool that was not removed by 300 mM NaCl treatment. In all cases, 5-LO was also found in cytosolic and non-nuclear membrane fractions. Indirect immunofluorescent microscopy confirmed the presence of abundant 5-LO within the nucleus with minimal extranuclear signal in most cells. However, a minority of cells, characterized by condensed chromatin, showed no nuclear-associated staining with increased cytoplasmic staining for 5-LO. This suggested that some of the cytosolic 5-LO found by cell fractionation resulted from these dividing cells. When the contribution from dividing cells was minimized, either by overnight serum deprivation or by isolating cytoplasts of nucleus-containing cells, 5-LO was prominent in the nuclear fraction but negligible in the cytosolic fraction. In contrast to this distribution in RBL cells, 5-LO in unstimulated human neutrophils was predominantly cytosolic, by both immunoblot and immunofluorescence analyses. In both RBL cells and human neutrophils, FLAP was localized at the nuclear membrane and the endoplasmic reticulum. These data provide the first evidence for the localization of 5-LO in unstimulated granulocytic cells. The finding that a substantial proportion of enzyme is localized within the nucleus of unstimulated RBL cells suggests potentially novel roles for 5-LO or its products within the nucleus.

A Chinese hamster fibroblast mutant defective in thrombin-induced signaling has a low level of phospholipase C-beta 1.

A mutant fibroblast, 2A4b, was isolated from the Chinese hamster lung cell line CCL39 by a previously described selection (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390) for cells deficient in thrombin-induced signaling. Although the antiporter activation by thrombin in 2A4b is only approximately 60% that in CCL39, the stimulation by serum is not significantly impaired, indicating that the defect in 2A4b lies upstream of the antiporter in the signaling pathway. The addition of thrombin to serum-starved 2A4b cells causes blunted responses both in production of inositol phosphates and in the cytosolic [Ca2+] transient, particularly when no Ca2+ is added to the external medium. The in vitro inositol phospholipid-specific phospholipase C (PLC) activity of 2A4b cytosol plus membrane extracts exceeds that in CCL39. However, immunoblots with antibodies to PLC isozymes show that although the levels of PLC-delta 1, PLC-gamma 1, and PLC-beta 3 are at least as great as those in CCL39, the amount of PLC-beta 1 in 2A4b is markedly deficient (< or = 10%). PLC-beta 1 is found primarily in the nucleus and in non-nuclear membranes of CCL39 and is proportionately low in these subcellular locations of 2A4b. Thrombin activation of phospholipases D and A2 is impaired in 2A4b. We postulate that the deficiency in PLC-beta 1 causes defective targeting of protein kinase C-alpha to specific membrane sites, which may be required for activation of these downstream phospholipases.

Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens.

Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters.

The nuclear localization of a putative neurotransmitter receptor.

A high affinity strychnine binding site has been identified within a membrane fraction prepared from partially purified rat brain nuclei. This interaction appears similar in its characteristics to that occurring in the non-nuclear membrane fraction which is thought to occur at the synaptic glycine receptor complex. Both the nuclear and non-nuclear membrane binding of tritiated strychnine is greater within the pons-medulla region than in the cerebral cortex. Nuclear membrane binding sites for dopamine, norepinephrine (beta-adrenergic), acetylcholine (muscarinic), GABA, and diazepam were not detected.

Chromatin influence on the function and formation of the nuclear envelope shown by laser-induced psoralen photoreaction.

Potorous tridactylis (PTK2) cells growing in culture were treated with psoralen derivatives and dividing cells were located by phase-contrast microscopy. Psoralens, light-sensitive DNA-photoadducting drugs, were reacted with mitotic chromosomes through exposure to 365-nm light from an argon laser microbeam system. It was found that following mitosis and photoreaction, cells without nuclear envelopes were produced when psoralen-treated cells received 60 light pulses over their entire chromosome complement. These 'non-nuclear membrane' cells were found to incorporate [3H]uridine and, to a lesser extent, [3H]thymidine by autoradiography. Reduction of the light exposure by half (30 near-u.v. pulses) over the entire chromosome complement in the presence of psoralen also produced non-nuclear-membrane cells as seen by light microscopy. Further examination of these cells (30 light pulses) by single-cell electron microscopy revealed that unlike the high light exposure (60 near-u.v. pulses), the low light dosage resulted in cells with membrane patches associated with their chromatin. Since neither actinomycin D nor cycloheximide impeded nuclear envelope reformation, the psoralen-DNA reaction is concluded to produce non-nuclear-membrane cells by a mechanism other than transcription or translation inhibition. The association of Golgi with areas of nuclear membrane patches gives indirect evidence of a possible Golgi contribution to the reformation of the nuclear envelope after mitosis. It is concluded that DNA plays a role in envelope reformation.

Pore patterns on nuclear membranes

The pore patterns on a large number of nuclear and one non-nuclear membrane have been examined. Important qualitative features of the arrangement of pores can be described by computing a statistical measure, the radial distribution function, of the pore patterns. This measure distinguishes three different pore patterns: 1. 1. Clumped patterns in which pores are found in clusters which are widely separated from one another. 2. 2. Patterns in which there is only a short range inhibitory region surrounding each pore with no evidence of a long range ordering. 3. 3. Patterns in which there is a local ordering in the region surrounding each pore, extending to several times the mean interpore spacing. Physical mechanisms which can lead to each of the observed patterns are discussed.