Liver Nuclei Research Articles

Macaque liver substrate for evaluating dense fine speckled-like patterns on HEp2010 cells

Antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) is instrumental in the evaluation of systemic autoimmune diseases (SADs). The dense fine speckled (DFS) ANA staining predominantly associates with anti-DFS70, an autoantibody that is thought to exclude the presence of SAD. However, the DFS pattern may mask the presence of other ANA patterns that may be clinically relevant. Our laboratory uses the HEp2010 substrate which contains both HEp2 and liver substates. The aim of this study was to determine whether negative liver nucleus immunofluorescence could exclude the presence of antibodies to extractable nuclear antigens (ENA) in sera with DFS-like patterns. One hundred consecutive sera samples suspicious for DFS pattern, along with 15 sera of control patterns (positive metaphase bars) were included in the study. Each sample was examined separately on HEp2010 (Euroimmun) and liver by two independent readers. Anti-DFS70 was assessed by line and chemiluminescent immunoassays. DFS-like sera were more likely to be liver nucleus-negative compared with control sera. Of the liver-negative sera, 61/64 (95.3%) were deemed anti-ENA negative. Using the liver substrate in the evaluation of anti-ENA had a sensitivity of 90.0% and a negative predictive value of 95.4%. In our cohort, concurrent evaluation of sera with the liver substrate helped rule out the presence of other anti-ENA. This technique may be a safeguard for DFS-like ANA patterns that may mask underlying anti-ENA on IIF.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

Evaluation of the Moonlighting Histone H3 Specific Protease (H3ase) Activity and the Dehydrogenase Activity of Glutamate Dehydrogenase (GDH).

The N-terminus of Histone H3 is proteolytically processed in aged chicken liver. A histone H3 N-terminus specific endopeptidase (named H3ase) has been purified from the nuclear extract of aged chicken liver. By sequencing and a series of biochemical methods including the demonstration of H3ase activity in bacterially expressed GDH, it was established that the H3ase activity was a moonlighting protease activity of glutamate dehydrogenase (GDH). However, the active site for the H3ase in the GDH remains elusive. Here, using cross-linking studies of the homogenously purified H3ase, we show that the GDH and the H3ase remain in the same native state. Further, the H3ase and GDH activities could be uncoupled by partial denaturation of GDH, suggesting strong evidence for the involvement of different active sites for GDH and H3ase activities. Through densitometry of the H3ase clipped H3 products, the H3ase activity was quantified and it was compared with the GDH activity of the chicken liver nuclear GDH. Furthermore, the H3ase mostly remained distributed in the perinuclear area as demonstrated by MNase digestion and immuno-localization of H3ase in chicken liver nuclei, as well as cultured mouse hepatocyte cells, suggesting that H3ase demonstrated regulated access to the chromatin. The present study thus broadly compares the H3ase and GDH activities of the chicken liver GDH.

An intrinsically disordered region controlling condensation of a circadian clock component and rhythmic transcription in the liver

Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBα, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription invivo. REV-ERBα condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBα. This work demonstrates physiological circadian protein condensates containing REV-ERBα whose IDR is required for hub formation and the control of rhythmic gene expression.

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

Perinatal Protein Restriction Impacts Nuclear O-GalNAc Glycosylation in Cells of Liver and Brain Structures of the Rat

BackgroundPost-translational modifications are key factors in the modulation of nuclear protein functions controlling cell physiology and an individual’s health. ObjectivesThis study examined the influence of protein restriction during the perinatal period on the nuclear O-N-acetylgalactosamine (O-GalNAc) glycosylation of cells from the liver and parts of the brain in the rat. MethodsPregnant Wistar rats were divided into 2 groups on day 14 of pregnancy and fed ad libitum 1 of 2 isocaloric diets containing 24% (well-fed) or 8% (protein-restricted diet) casein until the end of the experiment. Male pups were studied after weaning at 30 d of life. Animals and their organ/tissues (liver, cerebral cortex, cerebellum and hippocampus) were weighed. Cell nuclei were purified, and the presence in nucleus and cytoplasm of all factors required for the initiation of O-GalNAc glycan biosynthesis, i.e., the sugar donor (UDP-GalNAc), enzyme activity (ppGalNAc-transferase) and the glycosylation product (O-GalNAc glycans), were evaluated by western blotting, fluorescent microscopy, enzyme activity, enzyme-lectin sorbent assay and mass spectrometry. ResultsThe perinatal protein deficit reduced progeny weight, as well as the cerebral cortex and cerebellum weight. UDP-GalNAc levels in the cytoplasm and nuclei of the liver, the cerebral cortex, cerebellum, or hippocampus were not affected by the perinatal dietary protein deficits. However, this deficiency affected the ppGalNAc-transferase activity localized in the cerebral cortex and hippocampus cytoplasm as well as in the liver nucleus, thus reducing the “writing” ppGalNAc-transferase activity of O-GalNAc glycans. In addition, liver nucleoplasm from protein-restricted offspring revealed a significant reduction in the expression of O-GalNAc glycans on important nuclear proteins. ConclusionsOur results report an association between the consumption of a protein-restricted diet by the dam and her progeny with the modulation in the offspring’ liver nuclei O-GalNAc glycosylation, which may ultimately regulate nuclear protein functions.

Rats' liver morphological features under conditions of different supply with vitamins and minerals

The current practice of novel food safety assessment in the Russian Federation involves toxicological studies on the alimentary model of adaptation potential reduction of laboratory animals. Since vitamin and mineral deficiency can affect the size of structural elements of tissues, an objective estimation of the results obtained using this model is possible when determining the range of fluctuations of the studied morphometric parameters under conditions of different essential substances' supply, as well as under conditions of simulated toxic effects on the background of the corresponding supply. The purpose of the research was to investigate the morphological and morphometric features of the liver under the influence of reduced intake of vitamins and mineral elements in the combination with toxic effects of various nature, during growth and puberty of male Wistar rats. Material and methods. The article analyzed data of 4 model experiments on 140 animals that received semi-synthetic casein diet with different supply of vitamins B1, B2, B3, B6 and mineral elements Fe3+ and Mg2+, as well as data of 2 experiments on 180 animals with simulated toxic load of cadmium (Cd2+) salts and carbon tetrachloride. The animals were ~95 days old at the time of sampling, the duration of the experiments was ~65 days. For the analysis we used data on rats' body weight on the day of material sampling, absolute and relative liver weight, hepatocyte diameter, nucleus diameter and hepatocyte cytoplasm size in the central and peripheral zones of hepatic lobules. A total of 200 cells were analyzed in each group of animals. In accordance with the study design, all quantitative traits of the groups that received diets with an essential nutrient supply ranging from 75 to 2% were compared with the group that received a complete diet (100%). Results. Morphometric examination of hepatocytes revealed a linear decrease in the size of cell structural elements in the series of reducing the content of essential micronutrients in the diet. Under the conditions of 2-4% vitamin and mineral supply, cell and nucleus diameters as well as cytoplasm size were by ~16.8, 12.6 and 21.1% (p<0.05) lower respectively than in rats with optimal supply of these substances; under the conditions of 9-19% supply were by ~9.2, 9.7 and 8.7% lower (p<0.05); higher levels of supply caused reduction of hepatocyte, nucleus and cytoplasm sizes in a range not exceeding 5% (p>0.05). When comparing the size of hepatocytes of rats subjected to toxic load with the hepatocytes of rats referred to the reference standard, an increase in the size of hepatocytes under the action of carbon tetrachloride by 17.4% (p<0.05) on average and under the action of cadmium salts by 4.6% (p<0.05) was noted. Conclusion. Based on the analysis of liver morphological and morphometric studies' data, there were established sizes of hepatocytes structural elements in the rats kept on diets with decreasing supply with B group vitamins, iron and magnesium salts; the linear decrease in the sizes of structural elements of hepatocytes in the series of reduction of B group vitamins, iron and magnesium intake was revealed. Toxic exposures to carbon tetrachloride and cadmium salts against the background of a 19-30-75% supply with essential substances led to an increase in the hepatocytes size, the correlation between the degree of toxicant exposure and the supply level is not significant.

Mushroom Poisoning and Treatments

The poisoning mushroom can affect the human body and is even lethal. This review summarizes gastroenteritis, hemolytic and liver damage caused by poisoning mushroom, as well as therapy methods for a variety of toxins. The main symptoms of gastroenteritis include acute nausea, abdominal pain, and diarrhea. For that, the common treatments include vomiting, gastric lovage and catharsis. The manifestation of hemolytic contains interic, acute anemia, liver adds spleen enlargement. The main approaches used to alleviate the effects are cortisone and atrium biacrbonate. Moreover, serious anemic people can use adrenal cortical hormones or Transfusion Therapy. The symptoms of liver damage are quite similar to gastroenteritis and hemolytic, but the manifestation is instruction regarding form doctors and over-the counter medicines. With prompt treatment, most minor symptoms can be cured. However, if the toxin has damaged the endoplasmic reticulum from liver nucleus, it would be difficult for patients to recover. Future research may focuse on precise treatment methods of mushroom positioning. More efforts should be made to facilitate the awareness of people about poisoning mushrooms to prevent mushroom poisoning.

Accurate classification of liver nuclei based on AI models for diagnostic support of hepatocellular carcinoma

Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide. Early diagnosis is crucial to improve management and provide survival benefits to patients with HCC. Liver biopsy coupled to histopathology remains the “gold standard”. Visual inspection of biopsy images is a crucial examination to identify tissue and cellular alterations related to HCC, but it is limited by long timelines and by the variability in interpretation. Therefore, recent advances in digital microscopy and automatic image analysis have been developed for HCC classification, thus facilitating the final diagnosis.

Human IRP1 Translocates to the Nucleus in a Cell-Specific and Iron-Dependent Manner.

Iron regulatory protein 1 (IRP1) is a bifunctional protein with mutually exclusive RNA-binding or enzymatic activities that depend on the presence of a 4Fe-4S cluster. While IRP1 is a well-established cytosolic protein, work in a Drosophila model suggested that it may also exhibit nuclear localization. Herein, we addressed whether mammalian IRP1 can likewise translocate to the nucleus. We utilized primary cells and tissues from wild type and Irp1−/− mice, as well as human cell lines and tissue biopsy sections. IRP1 subcellular localization was analyzed by Western blotting, immunofluorescence and immunohistochemistry. We did not detect presence of nuclear IRP1 in wild type mouse embryonic fibroblasts (MEFs), primary hepatocytes or whole mouse liver. However, we observed IRP1-positive nuclei in human liver but not ovary sections. Biochemical fractionation studies revealed presence of IRP1 in the nucleus of human Huh7 and HepG2 hepatoma cells, but not HeLa cervical cancer cells. Importantly, nuclear IRP1 was only evident in iron-replete cells and disappeared following pharmacological iron chelation. These data provide the first experimental evidence for nuclear IRP1 expression in mammals, which appears to be species- and cell-specific. Furthermore, they suggest that the nuclear translocation of IRP1 is mediated by an iron-dependent mechanism.

Vitamin E performs antioxidant effect via PAP retrograde signaling pathway in Nile tilapia (Oreochromis niloticus)

PAP (3′-phosphoadenosine 5′-phosphate) is a ubiquitous phosphoric acid and a natural inhibitor of the XRN (5′-3'exoribonuclease) family. It was proved to enter the nucleus through the retrograde signaling pathway and inhibit XRN2 to prevent the degradation of miRNA precursors, thus promoting the anti-oxidation miRNA level in Arabidopsis thaliana. Vitamin E (tocopherol) was proved to promote the accumulation of PAP in the plant, which facilitates PAP into the nucleus to accomplish its antioxidant function. However, the relationship between VE and PAP in animals is unclear. To identify the relationship between VE and PAP and to uncover the function of PAP in fish, we investigated the performance of VE and PAP in Nile tilapia by comparing the antioxidant indicators (SOD, GSH-Px, and CAT), the Keap1-Nrf2 signaling pathway, and the miRNA expression profiles. Results showed that the antioxidant effect of VE and PAP showed similar character either in tilapia liver or in serum: the activities of GSH-Px and CAT of both groups were significantly increased (P < 0.05); the SOD activity of the VE group was significantly increased (P < 0.05), and although the result of the PAP group was not so significant (P > 0.05), PAP improved the SOD level, too. The two groups also showed similar character in the tilapia liver; both did not significantly increase the liver δ-VE content (P > 0.05). However, VE significantly increased the content of α-VE and γ-VE (P < 0.05), while the PAP group was insignificant (P > 0.05). Feed with VE and intraperitoneal injection of PAPs reagent both increased the PAP content in the liver of tilapia, and the effect of the VE group was more significant (P < 0.05) than that of the PAP group (P > 0.05). Both groups reduced the expression of Keap1 and Cullin3 genes and improved the level of HO-1 gene expression, with the improved miRNA level of Nrf2. As a logical result, they decreased the expression of XRN1 and XRN2. By profile sequencing, we further identified some antioxidant closely related miRNAs shared in the VE and PAP groups, including miR-30, miR-24, miR-19b, and miR-100. By comparing the regulating mechanism of VE and PAP of feed supply and intraperitoneal injection, we proved that VE and PAP were closely related in fish; VE promoted the gathering of PAP. The latter retrograded into the nucleus of the fish liver to inhibit the expression of XRN genes and to up-regulate antioxidant miRNA levels as it does in plants. Only the PAP can accomplish the antioxidant activities, while VE promotes the process. Our study laid the foundation for the application of PAP as a new antioxidant agent in fish farming and benefit a further understanding of the VE antioxidant function in fish.

Biomarker Responses, Gene Expression Alterations, and Histological Changes in Zebrafish (Danio rerio) After In Vivo Exposure to Polychlorinated Diphenyl Ethers.

Polychlorinated diphenyl ethers (PCDEs) have been detected in various aquatic matrices, which pose potential threats to aquatic ecosystem security. In this work, both micro and macro analysis methods were used to assess the toxicity of PCDEs to zebrafish. Results indicated that after in vivo PCDE exposure, the oxidative stress and related gene of Danio rerio were significantly changed. The higher concentration or longer exposure time could cause more severe oxidative stress in zebrafish tissues. Compared with among the five tested compounds, more obvious changes in the level of oxidative biomarkers of lower chlorinated PCDEs’ (4-mono-CDE and 4,4′-di-CDE) exposure groups were observed. The integrated biomarker response analysis and gene expression results also indicate a similar trend. Histopathological observation suggested that 4,4′-di-CDE could render liver nuclei enlargement and necrosis, hepatocyte vacuolation, and the development inhibition of ovarian cells. Transmission electron microscope photos showed that 4,4′-di-CDE caused organelle damage in the liver and ovary, including the rupture of the endoplasmic reticulum, swelling of mitochondria, and condensation of chromatin in the liver and mitochondria disappeared significantly in the ovary. The degree of damage is enhanced with the increasing exposure doses. In addition, PCDEs also significantly altered vitellogenin content and related gene (vtg1) expression, suggesting that PCDEs may be estrogen endocrine disruptors. Overall, these results provided some valuable toxicological data of PCDEs on aquatic species.

ScSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

Detection of superoxide radical in all biological systems by Thin Layer Chromatography

The study presents a new method that detects O2•−, via quantification of 2-hydroxyethidium (2-ΟΗ-Ε+) as low as ∼30 fmoles by High-Performance Thin Layer Chromatography (HPTLC). The method isolates 2-ΟΗ-Ε+ after its extraction by the anionic detergent SDS (at 18-fold higher than its CMC) together with certain organic/inorganic reagents, and its HPTLC-separation from di-ethidium (di-Ε+) and ethidium (Ε+). Quantification of 2-OH-E+ is based on its ex/em maxima at 290/540 nm, and of di-E+ and E+ at 295/545 nm. The major innovations of the present method are the development of protocols for (i) efficient extraction (by SDS) and (ii) sensitive quantification (by HPTLC) for 2-OH-E+ (as well as di-E+ and E+) from most biological systems (animals, plants, cells, subcellular compartments, fluids). The method extracts 2-ΟΗ-Ε+ (by neutralizing the strong binding between its quaternary N+ and negatively charged sites on phospholipids, DNA etc) together with free HE, while protects both from biological oxidases, and also extracts/quantifies total proteins (hydrophilic and hydrophobic) for expressing O2•− levels per protein quantity. The method also uses SDS (at 80-fold lower than its CMC) to extract/remove/wash 2-ΟΗ-Ε+ from cell/organelle exterior membrane sites, for more accurate internal content quantification. The new method is applied on indicative biological systems: (1) artificially stressed (mouse organs and liver mitochondria and nuclei, ±exposed to paraquat, a known O2•− generator), and (2) physiologically stressed (cauliflower plant, exposed to light/dark).

Study of dietary curcumin on the restorative effect of liver injury induced by carbon tetrachloride in common carp, Cyprinus carpio

Carbon tetrachloride (CCl4) can be decomposed into free radicals, which cause lipid peroxidation, thereby damaging liver cells, and resulting in the death of aquatic animals. The present study was to investigate the recovery effect and possible mechanism of dietary curcumin against liver injury in Cyprinus carpio induced by CCl4. The experiment was divided into four groups. Two control groups were fed with a basal feed (the negative control, P1 and the positive control, P2), and two treatment groups were fed with feeds supplemented with 120 and 240 mg kg−1 for the entire 10-week study duration, respectively (P3 and P4). At the end of the feeding period, the CCl4 solution (CCl4 was dissolved in olive oil at 30 %, 0.5 mL per 100 g body weight) was injected into the abdominal cavity of P1, P3 and P4 groups. And the P2 group was injected with olive oil in the same proportion. On days 2, 3, 5 and 7 after CCl4 challenge, samples were taken separately to investigate the effect of curcumin on the liver injury induced by CCl4 in fish. Results showed that plasma alanine transaminase (ALT)and aspartate aminotransferase (AST) avtivities of fish increased significantly, relative to pre-challenge levels, 2 day after CCl4-challenge in P1, P3 and P4 groups (P<0.05), and the avtivities of plasma ALT and AST in these groups decreased as challenge time from 2 day to 7day. Liver total superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and glutathione (GSH) content of fish in P1, P3 and P4 groups decreased firstly and then increased with increasing challenge time, however, the content of liver malondiadehyde (MDA) was contrary. Curcumin (120 mg kg-1 curcumin per feed) inhibited the damage of liver tissue structure caused by CCl4 and made liver tissue structure return to normal. Meanwhile, dietary curcumin supplementation could also increase the live nuclear factor crythroid 2-related factor 2 (Nrf2) mRNA level and Nrf2 protein level in the liver nucleus, especially 120 mg kg-1 feed. Overall, the results indicated that feed supplemented with curcumin could effectively protect the liver against CCl4 induced injury. And the 120 mg kg−1 curcumin group had the better restorative effect of liver injury compared with the high-dose group.

Metallobiochemistry of ultratrace levels of bismuth in the rat II. Interaction of 205+206Bi3+ with tissue, intracellular and molecular components

BackgroundKnowledge on Bi metabolism in laboratory animals refers to studies at “extreme” exposures, i.e. pharmacologically relevant high-doses (mg kg−1 b.w.) in relation to its medical use, or infinitesimal doses (pg kg−1b.w.) concerning radiobiology protection and radiotherapeutic purposes. There are no specific studies on metabolic patterns of environmental exposure doses (ultratrace level, μg kg−1 b.w.), becoming in this context Bi a “heavy metal fallen into oblivion”. We previously reported the results of the metabolic fate of ultratrace levels of Bi in the blood of rats [1]. In reference to the same study here we report the results of the retention and tissue binding of Bi with intracellular and molecular components. MethodsAnimals were intraperitoneally injected with 0.8 μg Bi kg−1 b.w. as 205+206Bi(NO)3, alone or in combination with 59Fe for the radiolabeling of iron proteins. The use of 205+206Bi radiotracer allowed the determination of Bi down to pg fg−1 in biological fluids, tissues, subcellular fractions, and biochemical components isolated by differential centrifugation, size exclusion chromatography, solvent extraction, precipitation, immunoprecipitation and dialysis. Main findingsAt 24 h post injection the kidney contained by far the highest Bi concentration (10 ng g−1 wt.w.) followed by the thymus, spleen, liver, thyroid, trachea, femur, lung, adrenal gland, stomach, duodenum and pancreas (0.1 to 1.3 ng g−1 wt.w.). Brain and testis showed smaller but consistently significant concentrations of the element (0.03 ng g−1 wt.w). Urine was the predominant route of excretion. Intracellularly, liver, kidney, spleen, testis, and brain cytosols displayed the highest percentages (35%–58%) of Bi of homogenates. Liver and testis nuclei were the organelles with the highest Bi content (24 % and 27 %). However, when the recovered Bi of the liver was recorded as percent of total recovered Bi divided by percent of total recovered protein the lysosomes showed the highest relative specific activity than in other fractions. In the brain subcellular fractions Bi was incorporated by neuro-structures with the protein and not lipidic fraction of the myelin retaining 18 % of Bi of the total homogenate. After the liver intra-subcellular fractionation: (i) 65 % of the nuclear Bi was associated with the protein fraction of the nuclear membranes and 35 % with the bulk chromatin bound to non-histone and DNA fractions; (ii) about 50 % of the mitochondrial Bi was associated with inner and outer membranes being the other half recovered in the intramitochondrial matrix; (iii) in microsomes Bi showed a high affinity (close to 90 %) for the membranous components (rough and smooth membranes); (iv) In the liver cytosol three pools of Bi-binding proteins (molecular size > 300 kDa, 70 kDa and 10 kDa) were observed with ferritin and metallothionein–like protein identified as Bi-binding biomolecules. Three similar protein pools were also observed in the kidney cytosol. However, the amount of Bi, calculated in percent of the total cytosolic Bi, were significantly different compared to the corresponding pools of the liver cytosol. ConclusionsAt the best of our knowledge the present paper represents the first in vivo study, on the basis of an environmental toxicology approach, aiming at describing retention and binding of Bi in the rat at tissue, intracellular and molecular levels.

Purification and partial characterization of vinculin from chicken liver nuclear extract

Vinculin is a well-known cytoskeletal protein and is a component of the integrin-mediated cell-matrix adhesion system. Recently, vinculin is also being reported from the nuclei from a number of organisms. However, there is no report yet on purification of nuclear vinculin from the native source of any organism. In the present study, by using western blotting, we show nuclear localization of vinculin in chicken liver. The chicken liver nuclear vinculin was purified to homogeneity and subsequently, the identity of vinculin was confirmed by peptide mass fingerprinting. Further, actin was co-immunoprecipitated with vinculin from chicken liver nuclear extract. Interestingly, the above immunoprecipitate (IP) demonstrated histone specific protease activity. Thus, the present study suggests plausible interaction of vinculin with actin and histone specific proteases in the chicken liver nuclei.