Inhibition of proliferation of primary avian fibroblasts through expression of histone H5 depends on the degree of phosphorylation of the protein.

To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.

Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B.

Experiments with antibodies induced by separated fragments 1-58 and 63-125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.

Effects of calorie restriction and aging on the expression of antioxidant enzymes and ubiquitin in the liver of Emory mice.

We studied the effect of age and calorie restriction on the expression of genes involved in antioxidant defenses in livers of young (4.5-6 months) and old (22 months) Emory mice fed a control (C) or restricted (R) diet. Specifically examined were catalase (CAT), glutathione peroxidase (Gpx), Cu/Zn and Mn superoxide dismutase (Cu/ZnSOD and MnSOD). As an indicator of oxidative damage to the tissues we measured lipid peroxidation. As indicators of oxidative stress we determined ubiquitin mRNA levels and endogenous high molecular weight (HMW) ubiquitin conjugates. Lower mRNA levels of ubiquitin (P < 0.05), CAT (P < 0.01) and Gpx (P < 0.01) were observed in tissues from young R versus C animals. The old C group had a lower CAT mRNA level (P < 0.0001) compared with young C. In the R group, age did not affect the CAT mRNA levels or Gpx mRNA levels; however, ubiquitin mRNA levels were higher (P < 0.05). No significant changes in Cu/Zn or MnSOD mRNA were observed with age or diet. Cu/ZnSOD protein levels were lower in the young R at 4.5 months (P < 0.05) than young C, and higher in the old R group versus old C (P < 0.05). CAT protein levels were higher in the old C versus old R (P < 0.05). Changes of HMW ubiquitin conjugates with age r diet were not significant. Of the four groups, the old R group showed the highest levels of lipid peroxidation.

Calorie restriction, stress and the ubiquitin-dependent pathway in mouse livers.

Calorie restriction (R) is the only known method to delay the aging process and extend mean and maximal lifespan in rodents. R has been shown to delay the age-related accumulation of damaged proteins and to protect organisms from various stresses which can produce damaged proteins. Such stresses include irradiation, heat shock, and oxidative stress. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) has been associated with the degradation of abnormal and/or damaged proteins. We examined the effect of diet and oxidative stress on activities of the UPP in supernatants from livers taken from 23-month-old Emory mice which had been exposed to an in-vivo injection of paraquat. Paraquat induces oxidative stress by generating superoxide radicals. In livers from non-stressed animals, steady-state levels of endogenous ubiquitin conjugates, de novo conjugate formation, and E1 and E2 activities were significantly lower in R animals than in control (C) animals. However, after exposure to paraquat, levels of endogenous ubiquitin conjugates were significantly higher in R versus C animals, and de novo conjugate formation and E1 and E2 activities in R animals rose to levels which were indistinguishable from levels of these activities noted in C animals. R was associated with an increased ability to degrade beta-lactoglobulin by the UPP after an oxidative stress was imposed. Ability to degrade beta-lactoglobulin by the C or R livers in non-stressed animals was not significantly different. Taken together, these data indicate that oxidative stress in R animals is associated with enhanced levels of ubiquitin conjugates and that this enhancement may be due to an increase in UPP activity. These data also indicate that the ability to form ubiquitin conjugates and the UPP system does not change with oxidative stress in C animals. The latter is consistent with prior reports that suggests that older C animals may already be in a state of enhanced oxidative stress and that activities of the UPP provide a sensitive indicator of levels of cellular redox status.

Aging, calorie restriction and ubiquitin-dependent proteolysis in the livers of Emory mice.

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade beta-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of beta-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).

The effects of aging and calorie restriction on plasma nutrient levels in male and female Emory mice.

We examined the effect of diet, age (4.5, 13 and 23 months), and sex on plasma levels of retinol, tocopherol, ascorbate, cholesterol, glucose and glycohemoglobin in male and female Emory mice which were fed control (C) and 50% calorie restricted (R) diets. Results showed that C fed animals tended to have higher levels of plasma ascorbate (50-71%), cholesterol (23-71%), glucose (38-81%) and glycohemoglobin (50%). However, these diet differences varied with the age and sex of the animals. Plasma retinol levels were lower only in R males vs. C males (50%). Novel sex-related differences in levels of plasma retinol (2-fold higher in C male mice than in C or R female mice) are described. Aging was associated with trends towards lower levels of plasma ascorbate (14-25%), glucose (34-36%) and glycohemoglobin (47-57%) from 4.5 to 23 months of age. However, these age differences depended upon the diet and sex of the animals. These data suggest that lower plasma levels of glucose, glycosylated hemoglobin and cholesterol may be causally related to the life extension noted in R animals since elevated levels of these moieties have been related to aging. Since oxidative stress is thought to be causally related to aging it appears unlikely that retinol, tocopherol and ascorbate are causally related to R-induced life-extension.

Conformational effects of histones H1 on DNA structure. Comparative study between H1-1, H1(0), H5 and sperm holothuria phi 0.

Interactions of mammalian histones, H1-1 and H1(0), phi 0 from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was phi 0 greater than H1-1 greater than H1(0). The binding of H1(0) to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1(0) with all polymers showed a strongly negative psi spectrum. Complexes of poly(dA-dT) and phi 0, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-like DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a psi spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with phi 0 showed spectral features characteristic of a mixture of a Z-like and a psi spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.

Cataract incidence and analysis of lens crystallins in the water-, urea- and SDS-soluble fractions of Emory mice fed a diet restricted by 40% in calories.

Restriction of dietary calorie intake is associated with life extension and with the delay of age-related disorders. Preliminary studies demonstrated that by feeding the Emory mouse a diet restricted by 21% in calories cataract and insolubilization of protein could also be delayed. To observe the effects of calorie restriction over prolonged portions of adulthood, Emory mice were fed the control diet (C) or a diet restricted by 40% in calories (R). Feeding the R diet was associated with delayed formation or progress of cataract over virtually the entire second half of life. At 11 months of age, bilateral grade 5 cataracts were present in 17% and 2% of C and R lenses, respectively. At 22 months of age, bilateral grade 5 cataracts were present in 90% and 18% of C and R lenses, respectively. The distribution of alpha-, beta-, and gamma- crystallins in the water-soluble, urea-soluble, and SDS-soluble fractions indicates more similarities than differences between C and R lenses with a specific grade of cataract or of a given age. However, there were significant and abrupt (after grade 4 cataract) losses of particular gamma-crystallins; gamma-crystallins which were not prominent at earlier stages became the major gamma-crystallin moieties. Losses of alpha-crystallins were also noted upon cataract formation or aging in most of the fractions. Aggregates including gamma- and alpha-crystallins also accumulate faster in the C group.

Serological detection of homologies of H1o with H5 and H1 histones.

Immunospecifically purified anti-chicken H5 antibodies caused bright staining of mouse liver nuclei in an indirect immunofluorescence assay. Analysis with the fluorescence-activated cell sorter showed that 95% of the nuclei were in the brightest category. Histones extracted from mouse liver nuclei, analyzed by sodium dodecyl sulfate-gel electrophoresis, presented an extra band with the same mobility as chick erythrocyte H5. Electrophoretic blots on nitrocellulose paper were treated with anti-H5 or anti-H1 antibodies and iodinated protein A. The affinity purified anti-H5 bound to chicken H5 and mouse H1o fractions only. Anti-calf H1 bound to calf, chicken, and mouse H1 proteins and to mouse H1o as well. Antibodies to purified subfractions of rat thymus H1 showed binding to both H1 and H1o. The results support the suggestions that H1o bears significant structural homology with H5 and H1.

Interactions of H1 and H5 histones with polynucleotides of B- and Z-DNA conformations.

Interactions of chicken H1 and H5 histones with poly(dA-dT), poly(dG-dC), and the Z-DNA structure brominated poly(dG-dC) were measured by a nitrocellulose filter binding assay and circular dichroism. At low protein:DNA ratios, both H1 and H5 bound more Z-DNA than B-DNA, and binding of Z-DNA was less sensitive to interference by an increase in ionic strength (to 600 mM NaCl). H5 histone bound a higher percentage of all three polynucleotides than did H1 and caused more profound CD spectral changes as well. For spectral studies, histones and DNA were mixed in 2.0 M NaCl and dialyzed stepwise to low ionic strength. Prepared in this way or by direct mixing in 150 mM NaCl, complexes made with right-handed poly(dG-dC) showed a deeply negative psi spectrum (deeper with H5 than with H1). Complexes of histone and Br-poly(dG-dC) showed a reduction in the characteristic Z-DNA spectral features, with H5 again having a greater effect. Complexes of poly(dA-dT) and H5, prepared by mixing them at a protein:DNA ratio of 0.5, displayed a distinctive spectrum that was not achieved with H1 even at higher protein:DNA ratios. It included a new negative band at 287 nm and a large positive band at 255 nm, giving the appearance of an inverted spectrum relative to spectra of various forms of B-DNA. These findings may reflect an ability of the different lysine-rich histones to cause varying conformational changes in the condensation of chromatin in DNA regions of highly biased base sequence.

Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes.

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.

Orientation of Arabidopsis thaliana KAT1 channel in the plasma membrane.

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K(+) channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K(+) channel, we have inserted a flag epitope in the NH(2) terminus or the S3-S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K(+) channel.

Serological homologies between H1 degrees and H5 include the carboxyl-terminal domain.

We have reported previously that antibodies to chicken H5 and antibodies to H1 both cross-react with mammalian H1 degree (Mura, C. V., and Stollar, B. D. (1981) J. Biol. Chem. 256, 9767-9769). The antigenic sites in H1 degree recognized by these antibodies were analyzed using immunoblotting. Peptides of H1 degree were prepared by partial digestion with acetic acid and tested for reactivity with: 1) antibodies induced by H5 alone, which reacted primarily with the central globular region of H5; 2) antibodies induced by H5 X RNA complexes, which reacted with this domain as well as the basic COOH-terminal domain; and 3) antiserum to calf thymus H1. Anti-H5 antibodies (anti-globular region) cross-reacted with H1 degree peptides that co-migrated with peptides of H5 that contain the globular region, but did not cross-react with H1. Anti-H5/RNA antibodies (anti-globular + anti-COOH-terminal) cross-reacted with these peptides and, in addition, with a lysine-rich H1 degree peptide that co-migrated with the basic COOH-terminal H5 peptide. This H1 degree peptide, but not the putative globular H1 degree peptides, was also recognized by an antiserum to calf H1 which was primarily reactive with the large, COOH-terminal N-bromosuccinimide fragment of calf H1. A weaker cross-reaction between this antiserum and the carboxyl-terminal domain of H5 could be visualized when large quantities of H5 were used in immunoblots. The results indicate that structural homologies between H5 and H1 degree extend beyond the globular region and into the lysine-rich carboxyl-terminal domain. Antigenic homologies between H1 degree and H1 are also at least partially localized in this domain. H1 degree is serologically intermediate between H5 and H1.

Purification and immunocytolocalization of protein phi 0 from sperm cells of the echinoderm Holothuria tubulosa.

Histones in chromatin from germ cells of the echinoderm Holothuria tubulosa are retained throughout spermatogenesis. However, some alterations occur in the histone complement of the mature sperm, including the presence of a germ-line-specific H1 subcomponent unusually rich in arginine, and the appearance of a basic component termed phi 0. Histones from ripe sperm have been extracted in a preparative scale to allow for isolation and purification of protein phi 0. Polyclonal antibodies against phi 0 have been produced and purified by affinity chromatography. The specificity of the antibodies to phi 0 has been assessed by enzyme-linked immunosorbent assays, competition experiments, and Western immunoblotting analysis. No cross-reactivity of the antibodies with the remainder histone fractions has been observed. Immunocytolocalization of protein phi 0 by immunogold labeling has revealed that this protein is essentially confined to chromatin from ripe sperm, whereas it is wholly absent from less advanced germ cell types. From these observations, together with biochemical studies previously reported, it is inferred that protein phi 0 may well be instrumental in the known chromatin transitions occurring in this organism during germ cell development.

Mapping of histone H5 sites on nucleosomes using immunoelectron microscopy.

The location of histone H5 on nucleosomes has been determined by binding anti-H5 antibodies to dinucleosomes, and recording the position of the bound IgG molecules using electron microscopy. Two types of antibody were employed, a total IgG fraction prepared from rabbits immunized with H5/RNA, which reacted to all three domains (NH2-terminal, central globular, and COOH-terminal) of H5, and an immunospecific subfraction which bound only to the central globular peptide. After reacting with dinucleosomes, both types of antibody were localized primarily in the linker DNA entry/exit region, but the whole antibody showed a much greater affinity for the linker DNA itself than did the antiglobular peptide antibody. These results provide direct support for the concept that H5, and by inference H1, is located at the linker DNA entry/exit site of the nucleosome, and further suggest that it is the central globular portion of the molecule that is most closely associated with this site. An interaction of one or both termini of H5 with the linker DNA is also indicated.

Characterization of the hemopoietic target cells for the avian leukemia virus E26.

The dual leukemogenic response, involving both the erythroid and myeloid hemopoietic systems in chickens infected with E26 virus, has previously been described (C. Moscovici, J. Samarut, L. Gazzolo, and M. G. Moscovici, 1981. Virology 113, 765-768; K. Radke, H. Beug, S. Kornfeld, and T. Graf, 1982. Cell 31, 643-653). Similarly, the in vitro response of the two lineages resulted in the concomitant transformation and proliferation of erythroblast and myeloblast leukemic cells. The present study, using embryonic tissues at very early stages of development, was valuable in implying that E26 target cells are recruited among uncommitted erythroid-myeloid stem cells as well as myeloid- or erythroid-committed progenitor cells. Therefore, E26 may be the first avian retrovirus capable of interacting with uncommitted hemopoietic precursor cells.

Distribution of antigenicity in chicken erythrocyte histone H5.

We have compared the inhibitory strengths of eight peptides from chicken histone H5 to that of the parent protein (189 residues) in complement fixation by guinea pig anti-H5 serum complexed with the homologous histone. Two precisely delineated regions (residues 59 to 65 and 94 to 99) and two less-defined regions (between 66 and 93; 100 and 189) are depicted. The sequences of particular consequences are quite conserved in avian histone H5 while the most variable N-terminal portion of 31 residues has no inhibitory effect.

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells.

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.