Rat Liver Nuclei Research Articles

Melting calorimetry of rat liver nuclei in the presence of magnesium ions

Differential scanning calorimetry was used to determine thermodynamic parameters of decondensation of intranuclear rat liver chromatin was induced by a decrease in the concentration of magnesium ions from 5 mM to 0 mM. The process of chromatin melting in the temperature range of 70-100°C occurs in the following order: melting of core-histones, melting of relaxed DNA, and melting of topologically constrained DNA. It was found that Tm and Д H of individual peaks also depend on the concentration of Mg2+ ions in the buffer. In nuclei with condensed chromatin, Mg2+ ions at a concentration of 5 mM increased significantly the Tm of core histones (by ~7°C), as compared to that in unfolded chromatin but at the same time lowered the Tm of nuclear DNA both in the relaxed and constrained state (by ~2.5°С and ~7.5°С, respectively). In the presence of Mg2+ ions, melting enthalpy for peaks increased significantly. At the same time, a decrease in molecular weights of intranuclear DNA levels out a stabilizing effect of Mg2+ ions on core histones. A rise in the concentration of Mg2+ ions above 5 mM leads to the appearance of a new peak with Tm above 100°С, which probably reflects the thermal behavior of some Mg-induced aggregates. Possible mechanisms underlying thermal behavior of chromatin inside the nucleus are discussed.

Melting Calorimetry of Rat Liver Nuclei in the Presence of Magnesium Ions

Melting Calorimetry of Rat Liver Nuclei in the Presence of Magnesium Ions

Nuclear-lipid-droplet proteome: carboxylesterase as a nuclear lipase involved in lipid-droplet homeostasis

Nuclear-lipid droplets (nLD)—a dynamic cellular organelle that stores neutral lipids, within the nucleus of eukaryotic cells—consists of a hydrophobic triacylglycerol –cholesterol-ester core enriched in oleic acid (OA) surrounded by a monolayer of polar lipids, cholesterol, and proteins. nLD are probably involved in nuclear-lipid homeostasis serving as an endonuclear buffer that provides or incorporates lipids and proteins participating in signaling pathways, as transcription factors and enzymes of lipid metabolism and nuclear processes. In the present work, we analyzed the nLD proteome and hypothesized that nLD-monolayer proteins could be involved in processes similar as the ones occurring in the cLD including lipid metabolism and other cellular functions. We evaluated the rat-liver–nLD proteome under physiological and nonpathological conditions by GeLC-MS2. Since isolated nLD are highly diluted, a protein-concentrating isolation protocol was designed. Thirty-five proteins were identified within the functional categories: cytoskeleton and structural, transcription and translation, histones, protein-folding and posttranslational modification, cellular proliferation and/or cancer, lipid metabolism, and transport. Purified nLD contained an enzyme from the lipid-metabolism pathway, carboxylesterase 1d (Ces1d/Ces3). Nuclear Carboxylesterase localization was confirmed by Western blotting. By in-silico analyses rat Ces1d/Ces3 secondary and tertiary structure predicted would be equivalent to human CES1. These results—the first nLD proteome—demonstrate that a tandem-GeLC-MS2-analysis protocol facilitates studies like these on rat-liver nuclei. A diversity of cellular-protein function was identified indicating the direct or indirect nLD participation and involving Ces1d/Ces3 in the LD-population homeostasis.

Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines.

We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased Tm of peaks II and III by ~5°C and decreased Tm of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (≤0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.

In vivo effect of borneol on rat hepatic CYP2B expression and activity

CYP2B subfamily accounts for 2–10% of total hepatic CYP450 enzymes and participate in the metabolism of around 8% of clinical drugs. Borneol has been widely used in traditional Chinese medicine for thousands of years. There are many studies about borneol-induced promoting penetration role for a number of drugs through various physiologic barriers, whereas there is no report involved the effect of borneol on hepatic CYP2B. The present work studied the in vivo effect of borneol on the expression and activity of rat hepatic CYP2B. The results indicated that the oral administration of borneol (33, 100 and 300 mg/kg/d) to rats for consecutive 7 days increased the hepatic CYP2B1/2 activity by 1.4-, 1.7- and 2.8-fold, hepatic CYP2B1 mRNA expression by 6.3-, 8.7- and 18.1-fold, and hepatic CYP2B1/2 protein expression by 1.2-, 1.9- and 2.6-fold, respectively compared to the control. Additionally, in the borneol pre-dosing (300 mg/kg/d for consecutive 7 days) rats, the increased Clint and decreased AUC0–24 of bupropion were observed as compared to the control. Moreover, there were no obvious effects on CAR protein level in rat liver microsome and nucleus following the borneol treatment. Taken together, our observations indicate that borneol is an in vivo inducer of rat hepatic CYP2B with different regulatory mechanism from phenobarbital-like inducers which caused CYP2B induction with CAR activation.

Extraction of histone H1 and decondensation of nuclear chromatin with various Mg-dependent organization levels under treatment with polyglutamic acid and distamycin.

Chromatin in rat liver nuclei under conditions of low ionic strength (20-25mM) and [Mg2+] from 2 to 5mM has a condensed structure (100-200nm globules) and gives the same CD signal (320-340nm) at interaction with the antibiotic distamycin A (DM). Reducing [Mg2+] to 1mM leads to chromatin decondensation to 30nm structures and increases the CD signal. Poly-L-glutamic acid (PG) at weight ratio PG/DNA= 6 and in the presence of 5mM Mg2+ extracts only about 1/8 of nuclear histone H1, preserving a condensed chromatin structure. Removal of about 1/4 of H1 at 3mM Mg2+ leads to chromatin decondensation to 30nm fibrils. Extraction of about half of histone H1 at [Mg2+] ≤ 2mM results in chromatin refolding to nucleosome fibrils. PG-decondensation leads to a significant increase in the CD signal. The main H1 extraction occurs in 1-2min, but at all Mg2+ concentrations the more slowly PG extracted fraction is found comprising 5-7% of nuclear H1. About 25% of leaving nuclear H1 can be extracted by PG in the presence of saturating DM concentration (molar DM/DNA= 0.1). H1 release depends significantly on the PG concentration. However, even at high weight ratio PG/DNA= 30 and DM/DNA= 0.1, about 5-10% of histone H1 remained in the nuclei. Decondensation of chromatin in the nucleus is not always proportional to the yield of extracted histone H1 and is weakened in the presence of positively charged DM or high concentrations of PG. Our results show that the interaction of DM with chromatin depends primarily on chromatin packaging, while PG extraction depends on [Mg2+] supporting this packaging.

FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro

In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100-200 nm; N-1) and partly decondensed (diameter of fibrils ~30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4-7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin structure, histone-chromatin binding strength, and concentration of DM.

Nuclear lipid droplets: A novel nuclear domain

We investigated nuclear neutral-lipid (NL) composition and organization, as NL may represent an alternative source for providing fatty acids and cholesterol (C) to membranes, signaling paths, and transcription factors in the nucleus. We show here that nuclear NL were organized into nonpolar domains in the form of nuclear-lipid droplets (nLD). By fluorescent confocal microscopy, representative nLD were observed in situ within the nuclei of rat hepatocytes in vivo and HepG2 cells, maintained under standard conditions in culture, and within nuclei isolated from rat liver. nLD were resistant to Triton X-100 and became stained with Sudan Red, OsO4, and BODIPY493/503. nLD and control cytosolic-lipid droplets (cLD) were isolated from rat-liver nuclei and from homogenates, respectively, by sucrose-gradient sedimentation. Lipids were extracted, separated by thin-layer chromatography, and quantified. nLD were composed of 37% lipids and 63% proteins. The nLD lipid composition was as follows: 19% triacylglycerols (TAG), 39% cholesteryl esters, 27% C, and 15% polar lipids; whereas the cLD composition contained different proportions of these same lipid classes, in particular 91% TAG. The TAG fatty acids from both lipid droplets were enriched in oleic, linoleic, and palmitic acids. The TAG from the nLD corresponded to a small pool, whereas the TAG from the cLD constituted the main cellular pool (at about 100% yield from the total homogenate). In conclusion, nLD are a domain within the nucleus where NL are stored and organized and may be involved in nuclear lipid homeostasis.

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1β and TNF-α) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of α2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a ‘sponge' for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR.

Editorial: Functional intracellular renin-angiotensin systems: potential for pathophysiology of disease

the renin-angiotensin system (RAS) is a coordinated hormonal cascade playing a major role in cardiovascular, renal, and adrenal homeostasis ([4][1]). Derangements in the RAS have been implicated in the pathogenesis of many disease states, including hypertension and of target organ damage related to

Influence of Distamycin, Chromomycin, and UV-irradiation on Extraction of Histone H1 from Rat Liver Nuclei by Polyglutamic Acid

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A(3) (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this difference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and ~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV-stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.

Influence of distamycin A on the methylation, extraction, and formation of UV-inducible DNA-protein cross links of histone H1 in the interphase nuclei of rat liver cells

Incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-(3)H]methionine ([(3)H] SAM) leads to incorporation of the radioactive label not only into core-histones H3 and H4, but also into linker histone H1. Addition of distamycine A to the incubation medium stimulates label incorporation into histone H1 ~ in 6 times and into histone H3 ~ in 2 times. The presence of distamycine facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases of UV-induced DNA-histone cross-links formation. These effects give evidence of weakening of H1-chromatin interaction by distamycin to be results of histone H1 position change relative to nucleosome and(or) disturbance of histones H1-H3 interactions so as these histones are exposed to additional methylation.

Carcinogenic heavy metals replace Ca 2+ for DNA binding and annealing activities of mono-ubiquitinated annexin A1 homodimer

Mono-ubiquitinated annexin A1 was purified from rat liver nuclei. The homodimer form of mono-ubiquitinated annexin A1 was able to unwind dsDNA in a Mg 2+- and ATP-dependent manner, and to anneal ssDNA in a Ca 2+-dependent manner. Phospholipids decreased the concentration of Ca 2+ required for maximal annealing activity. Heavy metals such as As 3+, Cr 6+, Pb 2+ and Cd 2+ substituted for Ca 2+ in the ssDNA binding and annealing activities of annexin A1. While these metals inhibited the unwinding of dsDNA by nuclear annexin A1 in the presence of Mg 2+ and ATP, they enhanced dsDNA-dependent ATPase activity of annexin A1. Heavy metals may have produced dsDNA, a substrate for the DNA unwinding reaction, via the DNA annealing reaction. DNA synthesomes were isolated from L5178Y tk(+/−) mouse lymphoma cells in exponential growth, and were found to contain helicase activities. The As 3+- or Cr 6+-induced increases in ssDNA binding activity of DNA synthesomes were reduced by a mono-specific anti-annexin A1 antibody, but not by anti-Ig antibody. Anti-annexin A1 antibody also blocked the inhibitory and stimulatory effects of As 3+ or Cr 6+ towards DNA unwinding and annealing activities of DNA synthesomes. Based on these observations, it can be concluded that the effects of heavy metals on DNA annealing and unwinding activities are mediated, at least in substantial part, through actions of the mono-ubiquitinated annexin A1 homodimer.

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague–Dawley rats (n=6 per group) were meal-fed (4h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P=0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9× higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.

Deleterious effects induced by oxidative stress in liver nuclei from rats receiving an alcohol-containing liquid diet

Highly purified rat-liver nuclei were previously shown to have nuclear ethanol (EtOH) metabolizing system able to bioactivate alcohol to acetaldehyde and 1-hydroxyethyl radicals. These reactive metabolites were able to covalently bind to nuclear proteins and lipids potentially being able to provoke oxidative stress of nuclear components. In this study, the above-mentioned possibility was explored. Sprague Dawley male rats (125-150 g) were fed a standard Lieber and De Carli liquid diet for 28 days. Controls were pair-fed with a diet, in which EtOH was isocalorically replaced with carbohydrate. The presence of a chlorzoxazone hydroxylase activity inducible by the repetitive EtOH drinking further suggested the presence of CYP2E1 in the highly purified nuclei. Nuclei from EtOH-drinking rats evidenced significantly increased susceptibility to a t-butyl hydroperoxide challenge as detected by chemiluminescence emission, increased formation of protein carbonyls, and decreased content of protein sulfhydryls. In contrast, no significant changes in the nuclear lipid hydroperoxides formation or even decreases in the 8-oxo-7,8-dihydro-2-deoxyguanosine were observed. No significant differences were observed in different parameters of the alkaline Comet assay. In immunohistochemical studies performed, no expression of p53 was observed in the livers of the animals under the experimental conditions tested. Since nuclear proteins and lipids are known to play a role in cell growth, differentiation, repair and signaling, their alterations by either oxidative stress, or by covalent binding might be of relevance to liver tumor promotion.

Glucose increases hepatic lipase expression in HepG2 liver cells through upregulation of upstream stimulatory factors 1 and 2

Elevated hepatic lipase (HL, also known as LIPC) expression is a key factor in the development of the atherogenic lipid profile in type 2 diabetes and insulin resistance. Recently, genetic screens revealed a possible association of type 2 diabetes and familial combined hyperlipidaemia with the USF1 gene. Therefore, we investigated the role of upstream stimulatory factors (USFs) in the regulation of HL. Levels of USF1, USF2 and HL were measured in HepG2 cells cultured in normal- or high-glucose medium (4.5 and 22.5 mmol/l, respectively) and in livers of streptozotocin-treated rats. Nuclear extracts of cells cultured in high glucose contained 2.5 +/- 0.5-fold more USF1 and 1.4 +/- 0.2-fold more USF2 protein than cells cultured in normal glucose (mean +/- SD, n = 3). This coincided with higher DNA binding of nuclear proteins to the USF consensus DNA binding site. Secretion of HL (2.9 +/- 0.5-fold), abundance of HL mRNA (1.5 +/- 0.2-fold) and HL (-685/+13) promoter activity (1.8 +/- 0.3-fold) increased in parallel. In chromatin immunoprecipitation assays, the proximal HL promoter region was immunoprecipitated with anti-USF1 and anti-USF2 antibodies. Co-transfection with USF1 or USF2 cDNA stimulated HL promoter activity 6- to 16-fold. USF and glucose responsiveness were significantly reduced by removal of the -310E-box from the HL promoter. Silencing of the USF1 gene by RNA interference reduced glucose responsiveness of the HL (-685/+13) promoter region by 50%. The hyperglycaemia in streptozotocin-treated rats was associated with similar increases in USF abundance in rat liver nuclei, but not with increased binding of USF to the rat Hl promoter region. Glucose increases HL expression in HepG2 cells via elevation of USF1 and USF2. This mechanism may contribute to the development of the dyslipidaemia that is typical of type 2 diabetes.

Comparative qualitative and quantitative analysis of scleroderma (systemic sclerosis) serologic immunoassays

The heterogeneity of autoantibody specificities occurring in sera from patients with systemic sclerosis (SSc) raised the necessity of developing various methodologies for their detection. A cohort of 150 SSc patients were selected and tested by Indirect Immunofluorescence (IIF), Counterimmunoelectrophoresis (CIE), Immunoblot (IB) using various extracts as antigenic source and RNA precipitation. By preparing a nuclear (IB-nuclear) and a metaphase chromosomal-enriched extract (IB-MC-pellet) from HeLa cells as well as a nucleolar (IB-nucleolar) and a histone (IB-histone) extract from rat liver nuclei, we assessed their sensitivity and specificity for anti-Topo I, anti-U 3RNP, anti-H 1, anti-snRNPs antibodies and ACA. IB-nuclear revealed the highest frequency of anti-Topo I antibodies, while CIE, IB-nucleolar and IB-MC-pellet, when compared to IB-nuclear showed a sensitivity of 89%, 87% and 95%, respectively. IB-MC-pellet was unique for ACA recognition, while IB-nucleolar and IB-MC-pellet showed excellent sensitivity for anti-U 3RNP and anti-H 1 antibody detection. We conclude that IB-nuclear is a highly sensitive system for anti-Topo I antibodies determination, but CIE reveals a good sensitivity to be used as a first screening test. IB-nucleolar or IB-MC-pellet are important techniques to detect the variety of antibodies to nucleolus and chromatin-related constituents. A novel specificity against a 28 kD nucleolar protein, non-associated with RNAs is also presented.

Thematic Review Series: Sphingolipids. Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.

Paradoxical effect of the long chain omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on nuclear transcription in vitro

Long chain unsaturated fatty acids are intimately involved in both the cause and prevention of various human diseases, e.g. obesity and diabetes, cardiovascular diseases, inflamation and allergy, and cancers including breast cancer. However, the long chain omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are believed to have protective effects against the above listed diseases. The mechanism(s) is not clear. But it is reasonable to believe that these beneficial effects are most likely through a common pathway deeply rooted at the gene level. This is to report the effect of EPA and DHA on nuclear transcription in vitro. We found that when EPA or DHA was added to the isolated rat liver nuclei, the nuclear RNA synthesis was stimulated over 2‐fold. This effect was determined to be a result of an increased rate of elongation of the nuclear RNA synthesis, and the rate at the initiation was not affected. Surprisingly, we found also that both EPA and DHA were able to strongly inhibit the enzyme activity of the isolated “free” nuclear RNA polymerase per se. We believe this is the first report on the paradoxical effect of EPA and DHA on the stimulation of the “engaged” and inhibition of the “free” nuclear RNA polymerase activity [Yu, FL. (1974) Two functional states of the RNA polymerases in the rat hepatic nuclear and nucleolar fractions. Nature 251, 344–346]. We hope that this finding will inspire further investigations into the mechanism of this paradoxical effect.