Nuclear Membrane Fractions Research Articles

Comparison of different methods for the isolation of Arabidopsis thaliana nuclear membranes

In animal and yeast system, subcellular fractionation has been widely used in studies of protein localization and organelle proteomics. Alternatively, it has not been an effective way of study in plant system because of some experimental limitations. The main aim of this study is to optimize subcellular and subnuclear fractionation of wild-type of Arabidopsis thaliana ecotype Colombia (Col-0) leaves by comparing three different methods to isolate nuclear membrane. In the study, we at first optimized nuclear washing steps to remove chloroplast contents from nuclear fractions. By optimizing speed of centrifugation and chemical component of the nuclear washing buffer, purified nuclear fractions was obtained. After measurement of protein amount for each fraction, purity of the fractions was analyzed by western blot assay with some specific cell compartment markers such as anti-Histone3 for nuclear fraction and anti-Rubisco for cytoplasmic fraction. Also, lactate dehydrogenase enzyme assay was used to confirm purity of the fractions. Then, subnuclear fractionation was done to isolate purified nuclear membrane. Three different methods were used to separate the nuclear membrane from whole purified nucleus. Nuclear compartment markers such as anti-Histone3 and anti-Fibrillarin was used in this step. In the study, Inner Nuclear Membrane protein (AtSUN2) was used as a nuclear membrane marker. All things considered, we conclude that the method with DNase digestion and high centrifugation speed (first method ) is a more effective way in separation of nuclear membrane fractions because the low centrifugal speed (second method) does not appear to be sufficient for separating the nuclear membrane, and the third method does not seem to be a very effective way as it requires a high centrifugal speed.

TLR2 activation induced by H. pylori LPS promotes the differential expression of claudin-4, -6, -7 and -9 via either STAT3 and ERK1/2 in AGS cells

Gastric carcinogenesis has been associated to H. pylori virulence factors that induce a chronic inflammation process. Lipopolysaccharides play a role in chronic inflammatory responses via TLR2- and TLR4-dependent signaling pathways. Similarly, cellular invasiveness, metastatic potential and prognosis are usually associated to claudin-4, -6, -7 and -9 expression in gastric carcinogenesis. Therefore, the aim of this study was to determine if H. pylori LPS exerts an influence on carcinogenesis-related claudin expression and if it was directly regulated through the TLR2 pathway. Human antrum gastric adenocarcinoma AGS cells exposed or not to H. pylori LPS were used. Polyclonal anti-claudin-4, -6, -7 and -9, anti-TLR2, anti-pERK1/2 as well as rabbit monoclonal anti-pNFκB p65 and mouse monoclonal anti-CdX2 were used. ERK1/2 inhibitor UO126 and STAT3 inhibitor Stattic were also used. Western blot, immunofluorescence and confocal experiments were performed in whole cells as well as total protein, nuclear and cell membrane fractions. The results showed that H. pylori LPS increased the expression of TLR2 in a time dependent bi-phasic manner (<12 and >12h exposure). Immunofluorescence using AGS monolayers corroborated the double phase TLR2 expression mainly on the cell membrane but a detectable signal was also determined in the cytoplasm of the cells. Activation of NFkB was downstream and depended on TLR2 expression as a statistically significant increase in pNFkB, that followed a pattern highly similar to the TLR2 expression was observed on the cell membrane fraction. The increase in TLR2 expression was accompanied by dramatically increased claudin-4 expression in cultures exposed from 30m to 8h to LPS. Increased expression of claudin-6, -7 and -9 also increases in >12h LPS exposure times. The increase in claudins expression was also dependent on NFkB activation. The results also showed an increase in pSTAT3 that followed a bi-phasic pattern that began 30min after stimulation and was compatible with the increase in TLR2 expression. The expression of the claudin-4 related CDX2 transcription factor did not followed the biphasic pattern. The results also showed that claudin-4 expression was STAT3 dependent whereas claudin-6, 7 and 9 expressions was ERK1/2 dependent. Our results suggest that H. pylori LPS induces TLR2 expression in the AGS cells, and that the longer the exposure to LPS, the greater the expression of TLR2 in the cell membrane. Consequently the expression of claudin-4, -6, -7 and -9 also increases.

Divergent roles of p120-catenin isoforms linked to altered cell viability, proliferation, and invasiveness in carcinogen-induced rat skin tumors.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) targets multiple organs for tumorigenesis in the rat, including the colon and the skin. PhIP-induced skin tumors were subjected to mutation screening, which identified genetic changes in Hras (7/40, 17.5%) and Tp53 (2/40, 5%), but not in Ctnnb1, a commonly mutated gene in PhIP-induced colon tumors. Despite the absence of Ctnnb1 mutations, β-catenin was overexpressed in nuclear and plasma membrane fractions from PhIP-induced skin tumors, coinciding with loss of p120-catenin from the plasma membrane, and the appearance of multiple p120-catenin-associated bands in the nuclear extracts. Real-time RT-PCR revealed that p120-catenin isoforms 1 and 4 were upregulated in PhIP-induced skin tumors, whereas p120-catenin isoform 3 was expressed uniformly, compared with adjacent normal-looking tissue. In human epidermoid carcinoma and colon cancer cells, transient transfection of p120-catenin isoform 1A enhanced the viability and cell invasion index, whereas transient transfection of p120-catenin isoform 4A increased cell viability and cell proliferation. Knockdown of p120-catenin revealed a corresponding reduction in the expression of β-catenin and a transcriptionally regulated target, Ccnd1/Cyclin D1. Co-immunoprecipitation experiments identified associations of β-catenin with p120-catenin isoforms in PhIP-induced skin tumors and human cancer cell lines. The results are discussed in the context of therapeutic strategies that might target different p120-catenin isoforms, providing an avenue to circumvent constitutively active β-catenin arising via distinct mechanisms in skin and colon cancer.

Cellular neurochemical characterization and subcellular localization of phospholipase C β1 in rat brain

The present study describes a complete and detailed neuroanatomical distribution map of the phospholipase C beta1 (PLCβ1) isoform along the adult rat neuraxis, and defines the phenotype of cells expressing PLCβ1, along with its subcellular localization in cortical neurons as assessed by double-immunofluorescence staining and confocal laser scanning. Immunohistochemical labeling revealed a considerable morphological heterogeneity among PLCβ1-positive cells in the cortex, even though there was a marked predominance of pyramidal morphologies. As an exception to the general non-matching distribution of GFAP and PLCβ1, a high degree of co-expression was observed in radial glia-like processes of the spinal cord white matter. In the somatosensory cortex, the proportion of GABAergic neurons co-stained with PLCβ1 was similar (around 2/3) in layers I, II–III, IV and VI, and considerably lower in layer V (around 2/5). Double immunofluorescence against PLCβ1 and nuclear speckle markers SC-35 and NeuN/Fox3 in isolated nuclei from the rat cortex showed a high overlap of both markers with PLCβ1 within the nuclear matrix. In contrast, there was no apparent co-localization with markers of the nuclear envelope and lamina. Finally, to assess whether the subcellular expression pattern of PLCβ1 involved specifically one of the two splice variants of PLCβ1, we carried out Western blot experiments in cortical subcellular fractions. Notably, PLCβ1a/1b ratios were statistically higher in the cytoplasm than in the nuclear and plasma membrane fractions. These results provide a deeper knowledge of the cellular distribution of the PLCβ1 isoform in different cell subtypes of the rat brain, and of its presence in the neuronal nuclear compartment.

Nuclear Na,K-ATPase plays an active role in Nucleoplasmic Calcium Homeostasis*

Na(+)/K(+)-ATPase, an integral membrane protein, has been studied for over a half century with respect to its transporter function in the plasma membrane, where it expels three Na(+) ions from the cell in exchange for two K(+) ions. In this study, we demonstrate a functioning Na(+)/K(+)-ATPase within HEK293 cell nuclei. This subcellular localization was confirmed by western blotting, ouabain-sensitive ATPase activity of the nuclear membrane fraction, immunocytochemistry and delivery of fluorescently tagged Na(+)/K(+)-ATPase α- and β-subunits. In addition, we observed an overlap between nuclear Na(+)/K(+)-ATPase and Na/Ca-exchanger (NCX) when nuclei were immunostained with commercially available Na(+)/K(+)-ATPase and NCX antibodies, suggesting a concerted physiological coupling between these transporters. In keeping with this, we observed an ATP-dependent, strophanthidin-sensitive Na(+) flux into the nuclear envelope (NE) lumen loaded with the Na-sensitive dye, CoroNa-Green. Analogous experiments using Fluo-5N, a low affinity Ca(2+) indicator, demonstrated a similar ATP-dependent and strophanthidin-sensitive Ca(2+) flux into the NE lumen. Our results reveal an intracellular physiological role for the coordinated efforts of the Na(+)/K(+)-ATPase and NCX to actively remove Ca(2+) from the nucleoplasm into the NE lumen (i.e. the nucleoplasmic reticulum).

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([3H] 4-dione) to [3H]dihydrotestosterone ([3H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [3H] 4-dione to [3H]DHT, homogenates of human prostate were incubated with [3H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [3H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [3H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [3H] 4-dione to [3H]DHT, showing IC50 values of 2 and 1.6 nM, respectively.

Glucocorticoid-induced fetal programming alters the functional complement of angiotensin receptor subtypes within the kidney.

We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT(1) receptors sensitive to losartan was 2-fold higher (67 ± 6% vs 27 ± 9%; P<0.01) in BMX compared with CON. In contrast, the proportion of AT(2) sites was only one third that of controls (BMX, 25 ± 11% vs CON, 78 ± 4%; P<0.01), with a similar reduction in sites sensitive to the Ang-(1-7) antagonist D-Ala7-Ang-(1-7) with BMX exposure. Functional studies revealed that Ang II stimulated ROS to a greater extent in BMX than in CON sheep (16 ± 3% vs 6 ± 4%; P<0.05); however, NO production to Ang II was attenuated in BMX (26 ± 7% vs 82 ± 14%; P<0.05). BMX exposure was also associated with a reduction in the Ang-(1-7) NO response (75 ± 8% vs 131 ± 26%; P<0.05). We conclude that altered expression of angiotensin receptor subtypes may be one mechanism whereby functional changes in NO- and ROS-dependent signaling pathways may favor the sustained increase in blood pressure evident in fetal programming.

Erratum to: Effects of alpha-linolenic acid vs. docosahexaenoic acid supply on the distribution of fatty acids among the rat cardiac subcellular membranes after a short- or long-term dietary exposure

Background Previous work showed that the functional cardiac effect of dietary alpha-linolenic acid (ALA) in rats requires a long feeding period (6 months), although a docosahexaenoic (DHA) acid-supply affects cardiac adrenergic response after 2 months. However, the total cardiac membrane n-3 polyunsaturated fatty acid (PUFA) composition remained unchanged after 2 months. This delay could be due to a specific reorganization of the different subcellular membrane PUFA profiles. This study was designed to investigate the evolution between 2 and 6 months of diet duration of the fatty acid profile in sarcolemmal (SL), mitochondrial (MI), nuclear (NU) and sarcoplasmic reticulum (SR) membrane fractions.

Steroid ‐ induced fetal programming alters angiotensin II receptor subtype expression in the sheep kidney

Antenatal exposure to glucocorticoids, as administered for preterm delivery, results in elevated mean arterial pressure (MAP) in offspring associated with reductions in glomerular filtration, renal plasma flow and nephron number. To determine whether renal angiotensin receptor changes accompany the alterations in hemodynamics in adulthood, fetal sheep (N=11) were administered doses of 0.17 mg/kg betamethasone (BMX) or saline (control) at 80–81 days gestation (dG) with full term delivery (~145 dG). Conscious MAP obtained in adult (1.8 years of age) BMX sheep was higher than controls (97 ± 3, N = 6 vs. 82 ± 2 mm Hg, N = 4; P&lt;0.01). Receptors in nuclear (NUC) and plasma membrane (PM) fractions were studied using 125I‐[Sar1, Thr8]‐Ang II and the antagonists Losartan (AT1) or PD123319 (AT2). BMX exposure increased the proportion of AT1 sites in cortex (NUC: 59 ± 4%; PM: 51 ± 5%) compared to controls (NUC: 28 ± 4%; PM: 25 ± 4%; P&lt;0.001). Moreover, BMX reduced cortical AT2 sites (NUC: 25 ± 14%; PM: 41 ± 10%) vs. controls (NUC: 84 ± 2%; PM: 88 ± 2%; P&lt;0.0001). In contrast to the cortex, medullary receptor subtypes were similar between groups. As we previously reported that AT1 receptor blockade reduces MAP in BMX‐treated adult sheep, an alteration in AT1: AT2 receptor ratio may be a key factor in steroid‐induced hypertension. Support: HL56973, HD047584, HD047584‐02S3 and HD017644.

Levels of Estrogen Receptors &amp;#945; and &amp;#946; in Frontal Cortex of Patients with Alzheimers Disease: Relationship to Mini-Mental State Examination Scores

Estrogen exerts beneficial effects on the brain throughout life. Studies demonstrate that estrogen is neuroprotective and that reduced brain estrogen activity may influence the clinical course of Alzheimer's disease (AD). Changes in levels of estrogen receptors have been detected in postmortem brain tissue of AD patients. Very little is known about the relationship between clinical stage and levels of estrogen receptors in postmortem brain. We hypothesized that estrogen receptor levels would be related to severity of cognitive impairment assessed proximate to death. Western blotting was used to quantify ER-alpha and ER-beta in nuclear, cytosolic, and crude membrane fractions of superior frontal cortex from 25 AD patients. Multiple linear regression analyses adjusted for age, sex, and education showed a significant linear relationship between Mini-Mental State Examination score (MMSE) and wild-type nuclear ER-alpha (â = 5.463, p = 0.03), but none between MMSE and wild-type nuclear ER-beta (â = 2.29, p = 0.36). We incidentally observed additional higher and lower molecular mass bands for ER-alpha in study subjects. Additional experiments performed on frontal cortex nuclear fractions prepared from subjects enrolled in a different study confirmed that these same bands are present in female and males with and without AD. Together our data show a relationship between wild-type ER-alpha and level of cognitive impairment in AD, and also suggest the possibility that variant isoforms of ER-alpha may be present in frontal cortex of patients with and without AD.

Functional Analysis of Nuclear Pore Complex Protein Nup62/p62 Using Monoclonal Antibodies

The nuclear pore complex (NPC) is an enormous structure embedded in the double membrane of the nuclear envelope that acts as a passageway for nucleocytoplasmic transport. The vertebrate NPC is comprised of about 30 unique proteins. Nup62/p62, a major component of the NPC, has been reported to interact directly with several nuclear transport factors, including importin-beta and NTF2. However, it has not been shown how the interaction of Nup62/p62 with transport factors is involved in nucleocytoplasmic transport. The present study reports on the preparation of monoclonal antibodies (MAbs) directed against human Nup62/p62 and a functional analysis of Nup62/p62 using antibodies in living cells. Hybridomas producing the antibodies were produced by the hybridization of mouse myeloma cells with medial iliac lymph node cells from an immunized rat. These MAbs specifically recognized Nup62/p62 as evidenced by immunoblotting analysis using a nuclear membrane fraction. In the immunostaining using MAbs, a punctuate nuclear rim staining pattern was observed. Moreover, cytoplasmic injected-anti-Nup62/p62 MAbs were rapidly targeted to the nuclear pore of cultured cells and some of them inhibited normal cell division, causing the formation of abnormal nuclei. The antibodies described in this study provide the means for immunochemical analyses of the NPC protein Nup62/p62 in mammalian cells, and represent useful molecular tools that should permit a better understanding of the biological roles and cellular dynamics of this protein in nucleocytoplasmic transport, cell division, and nuclear organization.

Specific Monoclonal Antibody Against the Nuclear Pore Complex Protein, Nup98

Nup98 is a component of nuclear pore complexes, which are large protein assemblies embedded in the nuclear envelope. Previous studies have shown that Nup98 interacts with several transport factors and plays a critical part in nuclear trafficking. However, the mechanism by which Nup98 contributes to nuclear trafficking is not clear. The present study reports on the preparation of a monoclonal antibody (MAb) directed against human Nup98. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. This antibody, MAb 2H10, specifically recognized Nup98, as evidenced by immunoblotting using a nuclear membrane fraction. In immunostaining using MAb 2H10, a punctuate nuclear rim staining pattern was observed. This MAb will be useful in immunoblotting and immunolocalization experiments in various cells and tissues, as well as further analyses of the biological function and cellular dynamics of this protein.

Presence of a Truncated Form of the Vitamin D Receptor (VDR) in a Strain of VDR-Knockout Mice

As part of our studies on the membrane-initiated actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [(3)H]-1alpha,25(OH)(2)D(3) in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5-50% of that seen in wild-type animals) specific binding of [(3)H]-1alpha,25(OH)(2)D(3) in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3-9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [(3)H]-1alpha,25(OH)(2)D(3) binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription.

Identification of Li+ binding sites and the effect of Li+ treatment on phospholipid composition in human neuroblastoma cells: a 7Li and 31P NMR study

Li(+) binding in subcellular fractions of human neuroblastoma SH-SY 5 Y cells was investigated using (7)Li NMR spin-lattice (T(1)) and spin-spin (T(2)) relaxation measurements, as the T(1)/T(2) ratio is a sensitive parameter of Li(+) binding. The majority of Li(+) binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li(+) binding constants and the values of the T(1)/T(2) ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by (31)P NMR spectroscopy the effects of chronic Li(+) treatment for 4--6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li(+) binding occurs predominantly to membrane domains, and that chronic Li(+) treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li(+) treatment of bipolar patients results in irregularities in Li(+) binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li(+) action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed.

Subcellular localization of estrogen receptor β in mouse hippocampus

While estrogen receptors have been known to represent estrogen-dependent transcription factors as part of the nuclear receptor family, a putative membrane-bound form of estrogen receptors has been suggested. Since estrogen receptor β (ERβ) is reportedly abundant in the hippocampus and other regions of the central nervous system, subcellular localization of ERβ in mouse hippocampus was investigated. ERβ was predominantly found in nuclear, synaptosomal and synaptic membrane fractions, particularly this last fraction. Immunocytochemical investigation using the NG108-15 neuroblastoma-glioma hybridoma cell line indicated that ERβ is predominantly localized in cell membranes and nuclei. These results suggest that ERβ localizes on synaptic membranes and may represent an important regulator of intracellular signal transduction from membrane to cytosol in hippocampal neurons.

Diminished Hepatic Response to Fasting/Refeeding and Liver X Receptor Agonists in Mice with Selective Deficiency of Sterol Regulatory Element-binding Protein-1c

Two treatments, fasting/refeeding and administration of liver X receptor (LXR) agonists, elevate the mRNA for sterol regulatory element-binding protein-1c (SREBP-1c) and enhance lipid synthesis in liver. These treatments do not affect the mRNA for SREBP-1a, an alternative transcript from the same gene. Through homologous recombination, we eliminated the exon encoding SREBP-1c from the mouse genome, leaving the SREBP-1a transcript intact. On a normal diet, livers of SREBP-1c(-/-) mice manifested reductions in multiple mRNAs encoding enzymes of fatty acid and triglyceride synthesis, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). In contrast, SREBP-1c(-/-) livers showed a compensatory increase in hepatic SREBP-2 mRNA, accompanied by increased mRNA levels for cholesterol biosynthetic enzymes. In fasted/refed animals, ACC and FAS mRNAs rose, but not to the same extent as in wild-type livers. The refeeding-induced increase in SREBP-1c(-/-) mice was greater than in mice lacking SREBP cleavage-activating protein (SCAP), in which all nuclear SREBPs are absent. Thus, SREBP-2 and/or SREBP-1a can substitute partially for SREBP-1c in permitting an insulin-mediated increase in ACC and FAS mRNAs. In contrast, mRNAs for several other lipogenic enzymes (glucose-6-phosphate dehydrogenase, malic enzyme, glycerol-3-phosphate acyltransferase, and stearoyl-CoA desaturase-1) showed a complete failure of the normal inductive response to refeeding, indicating specific reliance on SREBP-1c. Moreover, these mRNAs, as well as multiple other lipogenic mRNAs, showed a markedly blunted response to the LXR agonist T090137, indicating an essential role of SREBP-1c in the LXR response.

Fear Conditioning-Induced Alterations of Phospholipase C-β1a Protein Level and Enzyme Activity in Rat Hippocampal Formation and Medial Frontal Cortex

We investigated the effects of one-trial fear conditioning on phospholipase C-β1a catalytic activity and protein level in hippocampal formation and medial frontal cortex of untreated control rats and rats prenatally exposed to ethanol. One hour following fear conditioning of untreated control rats, phospholipase C-β1a protein level was increased in the hippocampal cytosolic fraction and decreased in the hippocampal membrane and cortical cytosolic and cortical membrane fractions. Twenty-four hours after fear conditioning, phospholipase C-β1a protein level was reduced in the hippocampal cytosolic fraction and elevated in the cortical nuclear fraction; in addition, 24 h after conditioning, phospholipase C-β1a activity in the cortical cytosolic fraction was increased. Rats that were exposed prenatally to ethanol displayed attenuated contextual fear conditioning, whereas conditioning to the acoustic-conditioned stimulus was not different from controls. In behavioral control (unconditioned) rats, fetal ethanol exposure was associated with reduced phospholipase C-β1a enzyme activity in the hippocampal nuclear, cortical cytosolic, and cortical membrane fractions and increased phospholipase C-β1a protein level in the hippocampal membrane and cortical cytosolic fractions. In certain cases, prenatal ethanol exposure modified the relationship between fear conditioning and changes in phospholipase C-β1a protein level and/or activity. The majority of these effects occurred 1 h, rather than 24 h, after fear conditioning. Multivariate analysis of variance revealed interactions between fear conditioning, subcellular fraction, and prenatal ethanol exposure for measures of phospholipase C-β1a protein level in hippocampal formation and phospholipase C-β1a enzyme activity in medial frontal cortex. In the majority of cases, fear conditioning-induced changes in hippocampal phospholipase C-β1a protein level were augmented in rats prenatally exposed to ethanol. In contrast, fear conditioning-induced changes in cortical phospholipase C-β1a activity were, often, in opposite directions in prenatal ethanol-exposed compared to diet control rats. We speculate that alterations in subcellular phospholipase C-β1a catalytic activity and protein level contribute to contextual fear conditioning and that learning deficits observed in rats exposed prenatally to ethanol result, in part, from dysfunctions in phospholipase C-β1a signal transduction.

Carotenoid incorporation into natural membranes from artificial carriers: liposomes and β-cyclodextrins

Liposomes and β-cyclodextrin (β-CD) have been used as carriers for the incorporation of three dietary carotenoids (β-carotene (BC), lutein (LUT) and canthaxanthin (CTX)) into plasma, mitochondrial, microsomal and nuclear membrane fractions from pig liver cells or the retinal epithelial cell line D407. The uptake dynamics of the carotenoids from the carriers to the organelle membranes and their incorporation yield (IY) was followed by incubations at pH 7.4 for up to 3 h. The mean IYs saturated between 0.1 and 0.9 after 10–30 min of incubation, depending on membrane characteristics (cholesterol to phospholipid ratio) and carotenoid specificity. Mitochondrial membranes (more fluid) favour the incorporation of BC (non-polar), while plasma membranes (more rigid) facilitate the incorporation of lutein, the most polar carotenoid. A high susceptibility of BC to degradation in the microsomal suspension was observed by parallel incubations with/without 2,6-di- t-buthyl- p-cresol (BHT) as antioxidant additive. The β-CD carrier showed to be more effective for the incorporation of lutein while BC was incorporated equally into natural membranes either from liposomes or from cyclodextrins. The presence of cytosol in the incubation mixture had no significant effects on the carotenoid incorporations.

Increased Activity of the Protein Kinase C-δ Holoenzyme in the Cytoplasmic Particulate Fraction Precedes the Activation of Caspases in Polyomavirus-Transformed pyF111 Rat Fibroblasts Exposed to Calphostin C or Topoisomerase-II Inhibitors

A caspase-mediated release of the 40-kDa catalytic fragment of the δ isoform (CF-δ) of protein kinase C (PKC-δ) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-δ, but cannot make the antiapoptotic Bcl-2 and Bcl-XL proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-δ activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101–108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737–743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-δ holoenzyme (holo-PKC-δ) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-δ fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-δ-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-δ. However, while calphostin C did not affect the δ-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the δ activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-δ activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of δ-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.

Localization of Functional Prostaglandin E2 Receptors EP3 and EP4 in the Nuclear Envelope

The effects of prostaglandin E2 are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP3 and the EP4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP3 and EP4. A perinuclear localization of EP3alpha and EP4 receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP3alpha and EP4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP3 receptors.