Antigen specificity of antihistone antibodies in systemic sclerosis

OBJECTIVES: The aim of the study was to determine the prevalence and clinical significance of antibodies to individual histone components in systemic sclerosis (SSc). METHODS: Serum samples from patients with limited cutaneous SSc (lSSc; n = 42) and diffuse cutaneous SSc (dSSc; n = 28) were examined for IgG and/or IgM antibodies to individual histone components and complexes by enzyme linked immunosorbent assay (ELISA). RESULTS: The level of IgG antibody to total histones was significantly higher in lSSc and dSSc than in normal controls. The level of IgM antibody to total histones was significantly higher in lSSc, but not in dSSc, than in normal controls. IgG antibody to total histones tended to be increased in dSSc when compared with that in lSSc. On the other hand, IgM antibody to total histones tended to be increased in lSSc when compared with that in dSSc. Although SSc showed various antihistone specificities, H2B, H2A-H2B, (H2A-H2B)-dsDNA were main antigens recognised by IgG antibodies in both lSSc and dSSc. Although IgM antibodies to H2B and H2A-H2B were also detected in both lSSc and dSSc, serum samples from lSSc patients exhibited highest IgM reactivity with H1. CONCLUSION: SSc may be included among conditions in which heterogeneous antihistone antibodies are produced. IgM antibodies to the most accessible histone H1 may be related to mild clinical features (lSSc) and IgG antibodies to the inner core molecules of native histone such as H2B or complexes including H2B may be associated with severe clinical features (dSSc) in Ssc.     

Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression

The chromatin elements targeted by the ATPdependent, Swi-Snf nucleosome-remodeling complex are unknown. To address this question, we generated mutations in yeast histone H2B that suppress phenotypes associated with the absence of Swi-Snf. Sin- (Swi-Snf-independent) mutations occur in residues involved in H2A-H2B dimer formation, dimer- tetramer association, and in the H2B N-terminus. The strongest and most pleiotropic Sin- mutation removed 20 amino acid residues from the H2B N-terminus. This mutation allowed active chromatin to be formed at the SUC2 locus in a snf5Delta mutant and resulted in hyperactivated levels of SUC2 mRNA under inducing conditions. Thus, the H2B N-terminus may be an important target of Swi-Snf in vivo. The GCN5 gene product, the catalytic subunit of several nuclear histone acetytransferase complexes that modify histone N-termini, was also found to act in conjunction with Swi-Snf. The phenotypes of double gcn5Deltasnf5Delta mutants suggest that histone acetylation may play both positive and negative roles in the activity of the Swi-Snf-remodeling factor.     

Rearrangement of the histone H2A C-terminal domain in the nucleosome

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.     

Interactions between arginine-rich histones and deoxyribonucleic acids. II. Circular dichroism

Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.     

Structural changes of soluble rat liver chromatin induced by the shift in pH from 7 to 9

Soluble rat liver chromatin was studied at pH 7 and at pH 9. In order to remove selectively non-histone components or non-histone components and histone H1, fractionation of chromatin was performed at pH 7 and pH 9 at different ionic strengths. The salt-dependent condensation of the fractionated chromatin was analysed in the electron microscope. There is no difference between the appearance of H1-depleted chromatin at poH 7 and pH 9. In H1-containing chromatin the shift from pH 7 to pH 9 leads to the following morphological changes: a) at very low ionic strength the nucleosomes unravel partially or totally and the zigzag-shaped fibres disappear in favour of beads-on-a-string; b) with increasing ionic strength the filaments condense into fibres, however, these fibres appear distorted and clearly less ordered than at pH 7. There is no indication of a release or displacement of histone H1. The pH-effect is completely reversible. The data suggest a pH-induced change in the mode of action of histone H1 in the formation of nucleosome beads and higher order chromatin structures.     

Ternary copper complexes and manganese (III) tetrakis(4-benzoic acid) porphyrin catalyze peroxynitrite-dependent nitration of aromatics

Peroxynitrite is a powerful oxidant formed in biological systems from the reaction of nitrogen monoxide and superoxide and is capable of nitrating phenols at neutral pH and ambient temperature. This peroxynitrite-mediated nitration is catalyzed by a number of Lewis acids, including CO2 and transition-metal ion complexes. Here we studied the effect of ternary copper-(II) complexes constituted by a 1,10-phenanthroline and an amino acid as ligands. All the complexes studied accelerate both the decomposition of peroxynitrite and its nitration of 4-hydroxyphenylacetic acid at pH > 7. The rate of these reactions depends on the copper complex concentration in a hyperbolic plus linear manner. The yield of nitrated products increases up to 2.6-fold with respect to proton-catalyzed nitration and has a dependency on the concentration of copper complexes which follows the same function as observed for the rate constants. The manganese porphyrin complex, Mn(III)tetrakis(4-benzoic acid)porphyrin [Mn(tbap)], also promoted peroxynitrite-mediated nitration with an even higher yield (4-fold increase) than the ternary copper complexes. At pH = 7.5 +/- 0.2 the catalytic behavior of the copper complexes can be linearly correlated with the pKa of the phenanthroline present as a ligand, implying that a peroxynitrite anion is coordinated to the copper ion prior to the nitration reaction. These observations may prove valuable to understand the biological effects of these transition-metal complexes (i.e., copper and manganese) that can mimic superoxide dismutase activity and, in the case of the ternary copper complexes, show antineoplastic activity.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.     

A cdc2-like kinase associated with commitment to division in Paramecium tetraurelia

Cell division in higher eukaryotes is mainly controlled by p34cdc2 or related kinases and by other components of these kinase complexes. We present evidence that cdc2-like kinases also occur in Paramecium. Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes. Only the less abundant peptide bound to p13suc1 from Schizosaccharomyces pombe. Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells. With this procedure, we demonstrated that the p13suc1-associated cdc2-like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium. Further, we show that Paramecium cdc2-like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.     

Histone neighbors in nuclei and extended chromatin

Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.     

The human histone gene cluster at the D6S105 locus

The sequences and organization of the histone genes in the histone gene cluster at the chromosomal marker D6S105 have been determined by analyzing the Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (YAC) 964f1. The insert of the YAC was subcloned in cosmids. In the established contig of the histone-gene-containing cosmids, 16 histone genes and 2 pseudogenes were identified: one H1 gene (H1.5), five H2A genes, four H2B genes and one pseudogene of H2B, three H3 genes, and three H4 genes plus one H4 pseudogene. The cluster extends about 80 kb with a nonordered arrangement of the histone genes. The dinucleotide repeat polymorphic marker D6S105 was localized at the telomeric end of this histone gene cluster. Almost all human histone genes isolated until now have been localized within this histone gene cluster and within the previously described region of histone genes, about 2 Mb telomeric of the newly described cluster or in a small group of histone genes on chromosome 1. We therefore conclude that the data presented here complete the set of human histone genes. This now allows the general organization of the human histone gene complement to be outlined on the basis of a compilation of all known histone gene clusters and solitary histone genes.     

Injury probability and risk in frontal crashes: effects of sorting techniques on priorities for offset testing

A hydrazinonicotinamide-functionalized cyclic platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist [HYNICtide, cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotina mido)hexanamide)))] was labeled with 99mTc using tricine and a series of imine-N-containing heterocycles as coligands. The imine-N-containing heterocycles include N-omega-Acetylhistamine (HIS-AC), N-(2-hydroxyethyl)isonicotinamide (ISONIC-HE), isonicotinic acid (ISONIC), isonicotinoyl-L-aspartic acid dimethyl ester (ISONIC-L-Asp-OMe2), 4-methyl-5-thiazoleethanol (MTE), nicotinic acid (NIC), 3-nitro-1,2,4-triazole (NTZ), 4-pyridylacetic acid (PA), 4-pyridineethanesulfonic acid (PES), and 3-pyridinesulfonic acid (PSA). The synthesis of these new ternary ligand [99mTc]HYNICtide complexes can be performed in one or two steps in high yield and high specific activity (>/=10 000 Ci/mmol HYNICtide). For example, the reaction of HYNICtide, [99mTc]pertechnetate, nicotinic acid, stannous chloride, and tricine at pH approximately 5 and 100 degreesC for 20 min results in the complex [99mTc(HYNICtide)(tricine)(NIC)] in >/=90% yield as determined by radio-HPLC. It was found that ternary ligand technetium complexes, [99mTc(HYNICtide)(tricine)(L)] (L = ISONIC, ISONIC-L-Asp-OMe2, ISONIC-HE, MTE, PA, PES, and PSA) are formed as equal mixtures of two isomeric forms. Complex [99mTc(HYNICtide)(tricine)(L)] (L = HIS-AC and NTZ) showed more than two well-resolved radiometric peaks at the retention times of interest, suggesting that they may have more than two forms in solution due to different bonding modalities of HIS-AC and NTZ. By a chirality experiment, it was found that the presence of two radiometric peaks is a result of the resolution of the two diastereomers which are formed by the combination of the chiral HYNICtide and the chiral technetium chelate. The formation of two diastereomers was also observed when a chiral imine-N-containing coligand was used for the radiolabeling of HYNIC-BA. The new ternary ligand [99mTc]HYNICtide complexes were found to be stable for up to 6 h in the reaction mixture. The high solution stability is attributed to their kinetic inertness. The composition of these complexes was determined to be 1:1:1:1 for Tc:HYNICtide:L:tricine (L = imine-N-containing heterocycles) through a series of mixed ligand experiments on the tracer (99mTc) level. The lipophilicity of the ternary ligand [99mTc]HYNICtide complexes can be systematically varied by the choice of polyaminocarboxylate and imine-N-containing coligands. Using the combination of tricine and an imine-N-containing coligand, HYNIC-derivatized peptides or other small molecules can be labeled with 99mTc in high specific activity and high stability for potential use as radiopharmaceuticals.     

Luminescence of Terbium Complexes Incorporated into Silica Matrix

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GdCl_3·3H_2O-18C6-C_2H_5OH体系(25℃)的相化学及固态配合物的合成与表征

采用半微量相平衡方法研究了 Gd Cl3· 3H2 O - 18C6 - C2 H5 OH三元体系 (2 5℃ )的溶解度 ,测定了各饱和溶液的折光率 ,考察了相平衡过程中水的行为。绘制了体系的溶解度图与饱和溶液折光率曲线。发现了两种未见文献报道的配合物 :3Gd Cl3· 18C6· 9H2 O· C2 H5 OH与 Gd Cl3· 18C6· 3H2 O。前者为固液异成分溶解的配合物 ,后者为固液同成分溶解的配合物。制备了固态配合物 ,通过化学分析、IR、DTG、TG以及 DSC研究了配合物的组成与性质 ,由 DSC得到了配合物若干分解步骤的焓变。用热化学方法求得了固态配合物 Gd Cl3· 18C6· 3H2 O(s)的标准生成焓     

The chlamydial EUO gene encodes a histone H1-specific protease

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.     

Removal of histone H1 from intact nuclei alters the digestion of nucleosome core DNA by staphylococcal nuclease

We have examined the gel profiles of staphylococcal nuclease digests of intact nuclei following different extents of removal of histone H1 by low pH. It was found that the submonomer fragment pattern (i.e. fragments less than 140 base pairs (bp) changed dramatically following removal of H1. The most striking feature of this change was a marked increase in the relative intensity of a band migrating at 102 +/- 4 bp when about 20-50% of the nuclear DNA is rendered acid soluble. All other submonomer bands decreased in relative intensity. There was no evidence for an approximately 100-bp repeat pattern accompanying the enhanced generation of the 102-bp fragment following H1 removal. This result, along with the comparisons of gel profiles for different extents of digestion, suggests that removal of histone H1 from nuclei results in an increased susceptibility of the DNA to staphylococcal nuclease at one or both ends of many of the core particles and that a strong block to further digestion occurs within these core particles resulting in the formation of a relatively stable 102-bp fragment.     

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.