Inhibition of AHNAK nucleoprotein 2 alleviates pulmonary fibrosis by downregulating the TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and advanced interstitial lung disease with poor prognosis. AHNAK nucleoprotein 2 (AHNAK2) is a macromolecular protein that is important for cell migration and muscle membrane repair. The protein acts via epithelial-mesenchymal transition (EMT), which is a key mechanism in the pathogenesis of IPF. However, very few studies have elucidated the effect of AHNAK2 in the development of IPF. Therefore, we aimed to determine the role of AHNAK2 in IPF development. METHODS: C57BL/6 mice were induced with bleomycin, while A549 and Beas-2b pulmonary epithelial cell lines were treated with TGF-ß1 to induce IPF model. The expression of AHNAK2 was detected using immunohistochemistry staining in vivo, and real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) in vitro. C57BL/6 mice were injected with adeno-associated virus (AAV)-sh NC or AAV-sh AHNAK2 and the pulmonary function and EMT marker expression were measured. The migratory abilities of the two transforming growth factor beta 1 (TGF-ß1)-induced cell lines were examined using wound-healing and Transwell assays after transfection with si-NC, si-AHNAK2-1 and -2. EMT marker expression was detected using RT-qPCR and WB. Smad3 and phosphorylated-Smad3 of the two cells were examined using WB. Following Smad3 inhibition by Smad3 phosphorylation inhibitor (SIS3), TGF-ß1-induced cell migration and EMT marker expression were evaluated again after different transfections. RESULTS: AHNAK2 expression was higher in the IPF model than in the normal model in vivo and in vitro. Partial inhibition of AHNAK2 suppressed the EMT process and improved pulmonary ventilation and compliance in the mouse model of IPF. Similarly, knockdown of AHNAK2 suppressed the migration of pulmonary epithelial cells and reversed EMT. Furthermore, Smad3 of the two TGF-ß1-induced cell lines was not activated when AHNAK2 was inhibited. When SIS3 inhibited the activation of Smad3, the suppression of AHNAK2 had no effect on A549 and Beas-2b, regardless of TGF-ß1 induction. CONCLUSIONS: Inhibition of AHNAK2 alleviates pulmonary fibrosis and partially reverses EMT by inhibiting the TGF-ß1/Smad3 signaling pathway. Therefore, AHNAK2 is a potential therapeutic target for IPF.

Nucleoprotein-enriched diet enhances protein synthesis pathway and satellite cell activation via ERK1/2 phosphorylation in unloaded rat muscles.

NEW FINDINGS: What is the central question of this study? The purpose of this study was to determine whether the nucleotides in a nucleoprotein-enriched diet could ameliorate the unloading-associated decrease in soleus muscle mass and fibre size. What is the main finding and its importance? The results indicate that the nucleotides in the nucleoprotein-enriched diet could ameliorate the unloading-associated decrease in type I fibre size and muscle mass, most probably owing to the activation of protein synthesis pathways and satellite cell proliferation and differentiation via ERK1/2 phosphorylation. Thus, nucleotide supplementation appears to be an effective countermeasure for muscle atrophy. ABSTRACT: Hindlimb unloading decreases both the protein synthesis pathway and satellite cell activation and results in muscle atrophy. Nucleotides are included in nucleoprotein and provide the benefits of increasing extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. ERK1/2 phosphorylation is also important in the activation of satellite cells, especially for myoblast proliferation and stimulating protein synthesis pathways. Therefore, we hypothesized that nucleotides in the nucleoproteins would ameliorate muscle atrophy by increasing the protein synthesis pathways and satellite cell activation during hindlimb unloading in rat soleus muscle. Twenty-four female Wistar rats were divided into four groups: control rats fed a basal diet without nucleoprotein (CON), control rats fed a nucleoprotein-enriched diet (CON+NP), hindlimb-unloaded rats fed a basal diet (HU) or hindlimb-unloaded rats fed a nucleoprotein-enriched diet (HU+NP). HU for 2 weeks resulted in reductions in phosphorylation of p70S6K and rpS6, the numbers of myoblast determination protein (MyoD)- and myogenin- positive nuclei, type I muscle fibre size and muscle mass. Both CON+NP and HU+NP rats showed an increase in ERK1/2, phosphorylation of p70S6K and rpS6, and the numbers of MyoD- and myogenin-positive nuclei compared with their basal diet groups. The NP diet also ameliorated the unloading-associated decrease in type I muscle fibre size and muscle mass. The results indicate that the nucleotides in the nucleoprotein-enriched diet could ameliorate the unloading-associated decrease in type I fibre size and muscle mass, most probably owing to the activation of protein synthesis pathways and satellite cell proliferation and differentiation via ERK1/2 phosphorylation. Thus, nucleotide supplementation appears to be an effective countermeasure for muscle atrophy.

L-arginine, an active component of salmon milt nucleoprotein, promotes thermotolerance via Sirtuin in Caenorhabditis elegans.

We previously showed that salmon milt nucleoprotein (NP) promotes thermotolerance in Caenorhabditis elegans; however, the active component and physiological mechanism of this effect has remained unclear. l-arginine (AR) is a major component of protamine and thus it has been proposed as the possible active component of NP. In this study, the viability of C. elegans treated with AR under heat stress was assessed and AR was shown to extend the survival term of the heat-stressed organisms. Additionally, AR was shown to restore the thrashing movement of the worms that is suppressed by heat stress. Treatment with AR was furthermore shown to promote thermotolerance in a DAF-16- and SIR-2.1-dependent manner, where DAF-16 and SIR-2.1 are homologs of FoxO and SirT1, respectively. Taken together, these data suggest that AR is one of the active components of NP and promotes thermotolerance via the activation of DAF-16 and SIR-2.1.

Protective Effects of Hydrolyzed Nucleoproteins from Salmon Milt against Ethanol-Induced Liver Injury in Rats.

Dietary nucleotides play a role in maintaining the immune responses of both animals and humans. Oral administration of nucleic acids from salmon milt have physiological functions in the cellular metabolism, proliferation, differentiation, and apoptosis of human small intestinal epithelial cells. In this study, we examined the effects of DNA-rich nucleic acids prepared from salmon milt (DNSM) on the development of liver fibrosis in an in vivo ethanol-carbon tetrachloride cirrhosis model. Plasma aspartate transaminase and alanine transaminase were significantly less active in the DNSM-treated group than in the ethanol plus carbon tetrachloride (CCl4)-treated group. Collagen accumulation in the liver and hepatic necrosis were observed histologically in ethanol plus CCl4-treated rats; however, DNSM-treatment fully protected rats against ethanol plus CCl4-induced liver fibrosis and necrosis. Furthermore, we examined whether DNSM had a preventive effect against alcohol-induced liver injury by regulating the cytochrome p450 2E1 (CYP2E1)-mediated oxidative stress pathway in an in vivo model. In this model, CYP2E1 activity in ethanol plus CCl4-treated rats increased significantly, but DNSM-treatment suppressed the enzyme's activity and reduced intracellular thiobarbituric acid reactive substances (TBARS) levels. Furthermore, the hepatocytes treated with 100 mM ethanol induced an increase in cell death and were not restored to the control levels when treated with DNSM, suggesting that digestive products of DNSM are effective for the prevention of alcohol-induced liver injury. Deoxyadenosine suppressed the ethanol-induced increase in cell death and increased the activity of alcohol dehydrogenase. These results suggest that DNSM treatment represents a novel tool for the prevention of alcohol-induced liver injury.

Effect of anti-Ebola virus monoclonal antibodies on endogenous antibody production in survivors of Ebola virus disease in the Democratic Republic of the Congo: an observational cohort study.

BACKGROUND: The use of specific anti-Ebola virus therapy, especially monoclonal antibodies, has improved survival in patients with Ebola virus disease. We aimed to assess the effect of monoclonal antibodies on anti-Ebola virus antibody responses in survivors of the 2018-20 Ebola outbreak in the Democratic Republic of the Congo. METHODS: In this observational prospective cohort study, participants were enrolled at three Ebola survivor clinics in Beni, Mangina, and Butembo (Democratic Republic of the Congo). Eligible children and adults notified as survivors of Ebola virus disease (ie, who had confirmed Ebola virus disease [RT-PCR positive in blood sample] and were subsequently declared recovered from the virus [RT-PCR negative in blood sample] with a certificate of recovery from Ebola virus disease issued by an Ebola treatment centre) during the 2018-20 Ebola virus disease outbreak were invited to participate in the study. Participants were recruited on discharge from Ebola treatment centres and followed up for 12-18 months depending on recruitment date. Routine follow-up assessments were done at 1, 3, 6, and 12-18 months after inclusion. We collected sociodemographic (age, sex, visit site), clinical (anti-Ebola virus drugs), and laboratory data (RT-PCR and Ct values). The primary outcome was the antibody concentrations against Ebola virus glycoprotein, nucleoprotein, and 40-kDa viral protein antigens over time assessed in all participants. Antibody concentrations were measured by the multiplex immunoassay, and the association between anti-Ebola virus antibody levels and the relevant exposures, such as anti-Ebola virus disease drugs (ansuvimab, REGN-EB3, ZMapp, or remdesivir), was assessed using both linear and logistic mixed regression models. This study is registered at ClinicalTrials.gov, NCT04409405. FINDINGS: Between April 16, 2020, and Oct 18, 2021, 1168 survivors were invited to participate in the Les Vainqueurs d'Ebola cohort study. 787 survivors were included in the study, of whom 358 had data available for antibody responses. 85 (24%) of 358 were seronegative for at least two Ebola virus antigens on discharge from the Ebola treatment centre. The antibody response over time fluctuated but a continuous decrease in an overall linear evolution was observed. Quantitative modelling showed a decrease in nucleoprotein, glycoprotein, and VP-40 antibody concentrations over time (p<0·0001) with the fastest decrease observed for glycoprotein. The probability of being seropositive for at least two antigens after 36 months was 53·6% (95% CI 51·6-55·6) for participants who received ansuvimab, 73·5% (71·5-75·5) for participants who received REGN-EB3, 76·8% (74·8-78·8) for participants who received remdesivir, and 78·5% (76·5-80·5) for participants who received ZMapp. INTERPRETATION: Almost a quarter of survivors were seronegative on discharge from the Ebola treatment centre and antibody concentrations decreased rapidly over time. These results indicate that monoclonal antibodies might negatively affect the production of anti-Ebola virus antibodies in survivors of Ebola virus disease which could increase the risk of reinfection or reactivation. FUNDING: The French National Agency for AIDS Research-Emergent Infectious Diseases-The French National Institute of Health and Medical Research, the French National Research Institute for Development, and the European and Developing Countries Clinical Trials Partnership. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

The nucleoprotein of influenza A virus inhibits the innate immune response by inducing mitophagy.

Mitophagy is a form of autophagy that plays a key role in maintaining the homeostasis of functional mitochondria in the cell. Viruses have evolved various strategies to manipulate mitophagy to escape host immune responses and promote virus replication. In this study, the nucleoprotein (NP) of H1N1 virus (PR8 strain) was identified as a regulator of mitophagy. We revealed that NP-mediated mitophagy leads to the degradation of the mitochondria-anchored protein MAVS, thereby blocking MAVS-mediated antiviral signaling and promoting virus replication. The NP-mediated mitophagy is dependent on the interaction of NP with MAVS and the cargo receptor TOLLIP. Moreover, Y313 of NP is a key residue for the MAVS-NP interaction and NP-mediated mitophagy. The NPY313F mutation significantly attenuates the virus-induced mitophagy and the virus replication in vitro and in vivo. Taken together, our findings uncover a novel mechanism by which the NP of influenza virus induces mitophagy to attenuate innate immunity.Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; ATG12: autophagy related 12; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; COX4/COXIV: cytochrome c oxidase subunit 4; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; EID50: 50% egg infective dose; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HEK: human embryonic kidney; hpi: hours post-infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MLD50: 50% mouse lethal dose; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NP: nucleoprotein; PB1: basic polymerase 1; RFP: red fluorescent protein; RIGI: RNA sensor RIG-I; RIGI-N: RIGI-CARD; SeV: Sendai virus; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOLLIP: toll interacting protein; TOMM20: translocase of outer mitochondrial membrane 20; TUBA: tubulin alpha; Vec: empty vector; vRNP: viral ribonucleoprotein.

The uptake and digestion of iodinated human serum albumin by macrophages in vitro.

Mouse peritoneal macrophages take up I*-HSA from their medium during in vitro cultivation. Conditions which promote I*-HSA uptake are the same as those which stimulate formation of pinocytic vesicles. Autoradiography of cells pulsed with (125)I-HSA showed that intracellular isotope is localized in perinuclear granules, or secondary lysosomes. Following a pulse of (125)I-HSA, intracellular radioactivity decreases and the amount of TCA-soluble isotope in the medium increases correspondingly. About 50% of the intracellular isotope is lost in 5 hr. The release of isotope from pulsed cells is not inhibited by parafluorophenylalanine, 2,4-dinitrophenol or by a reduction of the serum concentration of the medium. However, the processing of ingested (125)I-HSA is reversibly inhibited by reduced temperature. The TCA-soluble radioactive material excreted by pulsed macrophages was identified as monoiodotyrosine.

Nuclear actin and myosin as control elements in nucleocytoplasmic transport.

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.

The role of probiotic Lactobacillus acidophilus ATCC 4356 bacteriocin on effect of HBsu on planktonic cells and biofilm formation of Bacillus subtilis.

Bacillus subtilis is a Gram positive, aerobic and motile bacterium. Biofilm formation is an important feature of this bacterium which confers resistance to antimicrobial agents. The use of new antimicrobial reagents which eliminate biofilms are important and necessary. In this study, the effect of secondary metabolites (bacteriocin) from Lactobacillus acidophilus ATCC 4356 on Bacillus subtilis BM19 in the presence and absence of HBsu which is involved in the growth of planktonic cells and biofilm formation, is reported. HBsu nucleoprotein plays several roles in different processes of Bacillus subtilis cells such as replication, transcription, cell division, recombination and repair. In this study, for the first time, the effect of HBsu on biofilm formation is presented. RESULTS: In the absence of HBsu, purified bacteriocin from L. acidophilus ATCC 4356 was more effective in inhibiting growth of B. subtilis BM19 planktonic cells as well as biofilm formation. The presence of HBsu on the other hand led to increased biofilm formation.

Preventive effects of nucleoprotein supplementation combined with intermittent loading on capillary regression induced by hindlimb unloading in rat soleus muscle.

Physical inactivity leads to muscle atrophy and capillary regression in the skeletal muscle. Intermittent loading during hindlimb unloading attenuates the muscle atrophy, meanwhile the capillary regression in the skeletal muscle is not suppressed. Nucleoprotein has antioxidant capacity and may prevent capillary regression. Therefore, we assessed the combined effects of intermittent loading with nucleoprotein supplementation on capillary regression induced by hindlimb unloading. Five groups of rats were assigned: control (CON), 7 days hindlimb unloading (HU), HU plus nucleoprotein supplementation (HU + NP), intermittent loading during HU (HU + IL), and intermittent loading combined with nucleoprotein supplementation during HU (HU + IL + NP). Seven days HU resulted in decrease in capillary number-to-fiber number (C/F) ratio accompanied with disuse-associated changes in fetal liver kinase-1 (Flk-1), a proangiogenesis factor, and thrombospondin-1 (TSP-1), an antiangiogenesis factor, in the soleus muscle. In addition, citrate synthase (CS) activity was decreased and protein level of superoxide dismutase (SOD)-2 was increased. Neither nucleoprotein supplementation nor intermittent loading prevented the decrease in the C/F ratio, whereas nucleoprotein supplementation combined with intermittent loading prevented the regression of capillary during unloading. Moreover, the levels of Flk-1, TSP-1, and SOD-2 protein and the CS activity were maintained up to control levels. These results suggested that nucleoprotein supplementation combined with intermittent loading was effective to prevent capillary regression induced by muscle atrophy.

Nucleoprotein supplementation enhances the recovery of rat soleus mass with reloading after hindlimb unloading-induced atrophy via myonuclei accretion and increased protein synthesis.

Hindlimb unloading results in muscle atrophy and a period of reloading has been shown to partially recover the lost muscle mass. Two of the mechanisms involved in this recovery of muscle mass are the activation of protein synthesis pathways and an increase in myonuclei number. The additional myonuclei are provided by satellite cells that are activated by the mechanical stress associated with the reloading of the muscles and eventually incorporated into the muscle fibers. Amino acid supplementation with exercise also can increase skeletal muscle mass through enhancement of protein synthesis and nucleotide supplements can promote cell cycle activity. Therefore, we hypothesized that nucleoprotein supplementation, a combination of amino acids and nucleotides, would enhance the recovery of muscle mass to a greater extent than reloading alone after a period of unloading. Adult rats were assigned to 4 groups: control, hindlimb unloaded (HU; 14 days), reloaded (5 days) after hindlimb unloading (HUR), and reloaded after hindlimb unloading with nucleoprotein supplementation (HUR + NP). Compared with the HUR group, the HUR + NP group had larger soleus muscles and fiber cross-sectional areas, higher levels of phosphorylated rpS6, and higher numbers of myonuclei and myogenin-positive cells. These results suggest that nucleoprotein supplementation has a synergistic effect with reloading in recovering skeletal muscle properties after a period of unloading via rpS6 activation and satellite cell differentiation and incorporation into the muscle fibers. Therefore, this supplement may be an effective therapeutic regimen to include in rehabilitative strategies for a variety of muscle wasting conditions such as aging, cancer cachexia, muscular dystrophy, bed rest, and cast immobilization.

The pathogenesis of Ebola hemorrhagic fever.

Ebola virus causes lethal hemorrhagic disease in humans, yet there are still no satisfactory biological explanations to account for its extreme virulence. This review focuses on recent findings relevant to understanding the pathogenesis of Ebola virus infection and developing vaccines and effective therapy. The available data suggest that the envelope glycoprotein and the interaction of some viral proteins with the immune system are likely to play important roles in the extraordinary pathogenicity of this virus. There are also indications that genetically engineered vaccines, including plasmid DNA and viral vectors expressing Ebola virus proteins, and passive transfer of neutralizing antibodies could be feasible options for the control of Ebola virus-associated disease.

Methylation of chromatin in vitro.

The endogenous DNA methylase in nuclei isolated from growing mouse cells preferentially methylates DNA in micrococcal nuclease-resistant regions probably as a result of the location in these regions of the preponderance of hemimethylated sites. Added mouse ascites cell DNA methylase catalyses the methylation of exposed, nuclease-sensitive DNA in chromatin from growing or non-growing mouse or insect cells. The poor acceptor ability of nuclease-resistant regions in this situation is due to the presence of histone proteins which block de novo methylation. Transcriptionally active regions of chromatin are selectively methylated in vitro by either endogenous or added DNA methylase.

Nuclear ribonucleoproteins as inhibitors of mammalian RNA polymerase.

A ribonucleoprotein complex isolated from rabbit thymus nuclear lysates was found to be an inhibitor of DNA-dependent RNA polymerase II. The inhibition appeared to be of a competitive type and was completely reversed by high concentration of DNA. Highest inhibition was observed when enzyme and complex were preincubated before addition of DNA while there was little inhibition after enzyme had started synthesis on the DNA template. The RNA isolated from the complex was equally inhibitory and was a more effective inhibitor than either tRNA or rRNA.

A nucleoprotein-enriched diet suppresses dopaminergic neuronal cell loss and motor deficit in mice with MPTP-induced Parkinson's disease.

Parkinson's disease (PD) is an obstinate progressive neurodegenerative disease and characterized by locomotor impairment and dopaminergic neuronal degeneration in the substantia nigra pars compacta (SNc). We examined in here the dietary effect of nucleoprotein (NP) extracted from salmon soft roe on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected PD-like mice model to prevent the symptom as an alternative medicine. Male C57/BL6 mice were given either an artificially modified NP-free diet (NF) or NF supplied with 1.2% NP for 1 week. Then, mice were injected intraperitoneally four times with 20 mg/kg MPTP. Seven days later, locomotor activity was examined, and the brains were immunostained with tyrosine hydroxylase (TH) and Iba1 antibodies. Moreover, in situ detection of superoxide anion (O2(-)) and gene expression of mitochondrial electron transfer chain gene, Cox8b was evaluated in midbrains. NP-fed animals showed significantly reduced locomotor impairment and an increased number of TH-positive cells in the SNc compared with NF animals. The NP-fed animals also showed reduced lower levels of O2(-) and up-regulation of Cox8b levels and Iba1 immunoreactivity, suggesting that inflammation and oxidative stress were suppressed and mitochondrial impairment was relieved in these animals. Supplementation of the diet with NP may serve as a useful preventive measure to slow the onset of PD.

HIV-1 gp120 increases the permeability of rat brain endothelium cultures by a mechanism involving substance P.

OBJECTIVE: To analyse whether an HIV-1 envelope protein might play a role in damaging the blood-brain barrier as a fundamental step in the early invasion of the central nervous system by HIV-1. DESIGN: Analysis of permeability of rat brain endothelium cultures to albumin, to assess the functional integrity of the vascular component of the blood-brain barrier. METHODS: Rat brain endothelium cultures prepared by cerebral microvessels were exposed to recombinant gp120IIIB on microporous membranes and passage of biotin-labelled albumin was analysed. Scanning electron microscopy was used to analyse cell culture morphology. Some cultures were preincubated with N-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of nitric oxide synthase, or with spantide, a selective substance P antagonist. RESULTS: HIV-1 gp120 increased the permeability of rat brain endothelial cells to albumin in a dose-dependent manner. Scanning electron microscopy revealed profound gp120-induced alterations in cell morphology accounting for the increased permeability to macromolecules. These alterations were neutralized by anti-gp120 monoclonal antibody but not by isotype control antibody or L-NAME. By contrast, spantide and anti-substance P polyclonal antibody completely blocked the gp120-induced increase in albumin permeability. Control cultures exposed to measles virus nucleoprotein showed an increase in permeability that was not blocked by spantide. Brain endothelial cells, exposed to gp120, displayed cell surface immunoreactivity for substance P, suggesting that substance P is secreted by brain endothelium in response to gp120 stimulation and binds to brain endothelial cells through a receptor-mediated mechanism. CONCLUSIONS: These findings suggest a role for substance P in the gp120-induced increase in permeability of brain endothelium.

Properties of chromosomal proteins of human leukemic cells.

Purified chromatin isolated from lymphocytic cells derived from patients with acute leukemia, or other lymphoproliferative disorders has been compared with chromatin isolated from normal human lymphocytic cells by gel electrophoresis and differential gradient ultracentrifugation. Thermal denaturation studies showed higher Tm values for chromatin from leukemic cells, as compared to that of lymphocytic cells from normal donors or patients with infectious mononucleosis, reflecting the diverse complexity of these chromatins with respect to their varying chemical compositions. There are significant differences in the ratios of DNA:RNA:protein, as well as in the ratios of chromatin-associated histone and non-histone proteins; although chromatin-associated histones were more homogeneous than were the non-histone proteins, as adjudged by amino acid analyses and acrylamide gel electrophoresis. These differences in chromatin structure may relate to the differences in gene expression characteristic of these lymphocytic cells. The chromosomal acidic proteins isolated from the purified chromatin of human leukemic cells greatly stimulated the template activity of the chromatin in in vitro RNA synthesis. The non-histone proteins selectively interact with chromatins and influence the RNA polymerase reactions, indicating that there is selective tissue specificity of non-histone proteins.

Nuclear proteins damage by alkylating agents with different degrees of carcinogenicity.

We have tried to establish a correlation between the carcinogenic potency of two alkylating compounds and specific target sites in chromatin. We have therefore compared the nuclear metabolism of radioactively-labelled methylmethanesulfonate (MMS), a relatively weak carcinogen and N-methylnitrosourea (MNU), a highly potent carcinogen in cultured primary hepatocytes which have, high microsomal drug-metabolizing activity and in V79 Chinese hamster cells which have low microsomal enzymatic activity. The modification of specific amino acid residues in acid-soluble nuclear proteins (H) and non-histone nuclear proteins (NH) was studied after exposing the cells to various doses of alkylating agents overnight. We found that at all doses, mainly the cysteine (Cys), but also to a lower extent the histidine (His) residues are methylated in both H and NH protein fractions by MMS. At high doses of MMS, traces of methylarginine and methylated lysines could be detected. MNU predominantly methylates lysine and arginine residues, the former being found mostly in H, the latter in NH. Although both hepatocytes and V79 cells metabolized radioactively-labelled carcinogen, a higher percentage of counts were incorporated by the hepatocytes; 'unusually' methylated amino acids were detectable in the hepatocyte proteins with relatively low doses of the alkylating agents but not in V79 cells. In the presence of exogenous microsomes, during exposure of V79 cells to the alkylating agents, the amount of amino acid methylation is qualitatively and quantitatively similar to that found in hepatocytes. Our data suggest a specific mechanism of protein methylation, at the level of target amino acids, for carcinogens with different potencies similar to what has been found for DNA bases. A component of the microsomal fraction (S9) may be able to enhance this effect.

Inhibition of hepatic extramedullary haemopoiesis by nucleoprotein of heterologous rotavirus strain in infant mice.

Infant mice (NMRI strain) showed the inhibition of hepatic extramedullary haemopoiesis by oral inoculation of a 100 ID50 dose of EB rotavirus and nucleoprotein of SA-11 rotavirus (serotype 3). The extramedullary haemopoiesis was observed by oral inoculation of surface protein VP7 of SA-11 rotavirus and in control (placebo administered) mice.

[Biologically active ribonucleoproteins of cells infected with fowl plague virus]. / Biologicheski aktivnye ribonukleoproteidy kletok, zarazhennykh virusom chumy ptits.

The results of studies of physico-chemical and biological properties of virus-specific ribonucleoproteins (RNP) in influenza infection are presented. Particular attention is given to the infectious properties of RNP. The earliest infectivity was found to be associated with RNP structures sedimenting from nuclear extract in a zone of 30-40S.