Isolation and physico-chemical properties of native histone complexes: dimer (H2-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2

The paper is concerned with the isolation of the native histone complexes: dimer (H2A-H2B), tetramer (H3-H4)2 and octamer (H3-H4-H2A-H2B)2 from the calf thymus chromatin under soft conditions (hydroxyl apatite) fractionation with the subsequent gel filtration). Parameters of hydroxyl apatite saturation with chromatin are determined. The complexes obtained are free of DNA and nonhistone proteins. Absorption spectra parameters, quantum efficiencies and fluorescence spectra typical of the corresponding histone oligomers are established. Comparison of free tyrosine fluorescence spectra with histone tyrosyl ones revealed a long-wave shift in the latter.     

Spatial organization of the (H3-H4-H2A-H2B)2 histone octamer

Structure of the (H2A-H2B-H3-H4)2 histone octamer isolated from calf thymus chromatin at ionic strength 0.1 to 4.0 M NaCl, pH 7.6, was studied spectrofluorometrically. Sensitivity of lambda max tyrosine fluorescence position to structural changes of histone oligomers and to the processes of their association was shown. It were detect two ranges of cooperative changes in histone optical parameters at 0.6-1.4 M NaCl (transition I) and at 2.4-3.4 M NaCl (transition II): Transition I corresponds to the formation of equilibrium system (hexamer) + (dimer) in equilibrium octamer. Transition II corresponds to the structural changes of the histone octamer. Thus, fluorescence anisotropy increases, lambda max for fluorescence spectrum is shifted to the longer wavelengths, contributions of two components to fluorescence decay change, a fraction of fluorescence accessible to the quenching by I- decreases. Histone octamer formation is characterized by making specific contacts between the (H2A-H2B) dimer and (H3-H4)2 tetramer. These contacts are realized at gradual changing of ionic strengths (by dialysis). In the case of abrupt local changes of the environment the process is irreversibly shifted to formation of unspecific high molecular aggregates. The important function role for energetically degenerated states of histone oligomers, energy barriers between which can be overcome by changing total conditions of histone microenvironment in chromatin is discussed.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

Synthesis of [6″-H]-, (6″-H)- and (2-H)-maltotriose

To prepare labeled precursors for biosynthetic studies, methods for the specific introduction of tritium and deuterium into the reducing and the terminal glucose unit of maltotriose were developed. Thus [6″-³H]- and (6″-²H)-maltotriose (17) and (18) were prepared via selective methoxytritylation, deprotection and subsequent modified Pfitzner-Moffatt oxidation, followed by reduction with sodium borotritiide or sodium borodeuteride, respectively. A simple two step procedure utilizing the Lobry de Bruyn/van Ekenstein transformation gave (2-²H)maltotriose (20).     

Fe^2＋-H2O2体系降解壳聚糖

Fe2 -H2O2体系能够有效地降解壳聚糖,反应介质的pH值、反应时间、反应温度、Fe2 浓度及H2O2浓度等实验因素对壳聚糖的降解效果都有程度不同的影响,其中以反应介质的pH值和H2O2浓度对降解反应的影响为最大.在pH值为3～5时Fe2 -H2O2体系降解壳聚糖的活性最高.适当增大H2O2的用量可以增大壳聚糖的降解程度,但当其用量增大至一定程度后,壳聚糖降解产物分子量的下降趋势明显变缓.合理的Fe2 -H2O2体系降解壳聚糖的实验条件为:介质pH值3～5;温度,室温;时间60～90 min;壳聚糖:H2O2:Fe2 =240:12～24:1～2(摩尔比).     

Immune Checkpoints of the B7 Family. Part 1. General Characteristics and First Representatives: B7-1, B7-2, B7-H1, B7-H2, and B7-DC

Russian Journal of Bioorganic Chemistry - T-cell response along with humoral response compose the basis of acquired immunity. Effective activation of T lymphocytes requires at least two signals....     

Tissue-Expressed B7-H1 Critically Controls Intestinal Inflammation

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A rapid method of preparing the (H3-H4-H2A-H2b)(2) histone octamer in large quantities]

A simple and fast method for isolation of large amounts of the histone octamer (H2A-H2B-H3-H4)2 is proposed. This method is based on chromatin adsorption by hydroxyapatite with subsequent extraction of the histone octamer with 50 mM sodium-phosphate buffer containing 4 M NaCl pH 8.0. It was shown that the properties of the histone octamer isolated by this extractive procedure are identical with those of the histone octamer obtained by elution on a Sephadex G-100 column. The histone tetramer (H3-H4)2 and dimer (H2A-H2B) were obtained after gel filtration on Sephadex G-100 in 50 mM sodium-acetate (pH 5.6).     

Dual activity of the H1-H2 domain of the (Na(+)+K+)-ATPase

(Na⁺+K⁺)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the α-subunit of the (Na⁺+K⁺)-ATPase is one of the digitalis drug interaction sites of the enzyme. Despite the extensive studies of the inhibitory effect of digitalis on the (Na⁺+K⁺)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na⁺+K⁺)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na⁺+K⁺)-ATPase even in the presence of SSA78. These results suggest that the H1-H2 domain of the (Na⁺+K⁺)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na⁺+K⁺)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis or ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure-function relationships of the (Na⁺+K⁺)-ATPase and the biological processes mediated by the enzyme.     

Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)₂ tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)₂ tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)₂ tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.     

Immune Checkpoints of the B7 Family. Part 2. Representatives of the B7 Family B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, and ILDR2 and Their Receptors

Russian Journal of Bioorganic Chemistry - Immune checkpoints regulate polarity, strength, and termination of the immune response. The leading roles in these processes are played by molecules of the...     

rNM23-H1/NDPK-A中试纯化工艺研究

为比较3种DEAE填料对rNM23-H1/NDPK-A的纯化效果,利用同一批中试发酵样品在相同的条件下进行离子交换层析,分别收集P0．2和P1．0两个洗脱峰。通过对洗脱峰中蛋白质含量、目标蛋白相对含量以及酶比活的测定,计算得出Matrex Cellufine A-200,DEAE Sephadex A-25,Macro-Prep DEAE Support三种填料相对应的NDPK－A得率及纯化倍数分别为74．5％、40．8％、92．6％、2．4、1．9、3．1倍。综合分析表明Macro-Prep DEAE Support填料对rNM23-H1/NDPK-A的纯化效果最好。     

Post-Activation Brain Warming: A 1-H MRS Thermometry Study

PurposeTemperature plays a fundamental role for the proper functioning of the brain. However, there are only fragmentary data on brain temperature (T_br) and its regulation under different physiological conditions.MethodsWe studied T_br in the visual cortex of 20 normal subjects serially with a wide temporal window under different states including rest, activation and recovery by a visual stimulation-Magnetic Resonance Spectroscopy Thermometry combined approach. We also studied T_br in a control region, the centrum semiovale, under the same conditions.ResultsVisual cortex mean baseline T_br was higher than mean body temperature (37.38 vs 36.60, P<0.001). During activation T_br remained unchanged at first and then showed a small decrease (-0.20 C°) around the baseline value. After the end of activation T_br increased consistently (+0.60 C°) and then returned to baseline values after some minutes. Centrum semiovale T_br remained unchanged through rest, visual stimulation and recovery.ConclusionThese findings have several implications, among them that neuronal firing itself is not a major source of heat release in the brain and that there is an aftermath of brain activation that lasts minutes before returning to baseline conditions.     

Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis

 The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5′ to nm23-H1 and another 3′ to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed. Received: 5 May 1997 / Accepted: 2 June 1997     

In vitro induction of H1-H1 histone cross-linking by adenosine diphosphate-ribose polymers

It is well-known that H1-H1 interactions are very important for the induction of 30 nm chromatin fiber and that, among all posttranslational modifications, poly(ADP-ribosyl)ation is one of those capable of modifying chromatin structure, mainly through H1 histone. As this protein can undergo both covalent and noncovalent modifications by poly(ADP-ribosyl)ation, our aim was to investigate whether and how ADP-ribose polymers, by themselves, are able to affect the formation of H1-H1 oligomers, which are normally present in a condensed chromatin structure. The results obtained in our in vitro experimental system indicate that ADP-ribose polymers are involved in chromatin decondensation. This conclusion was reached as the result of two different observations: (a) H1 histone molecules can be hosted in clusters on ADP-ribose polymers, as shown by their ability to be chemically cross-linked, and (b) H1 histone has a higher affinity for ADP-ribose polymers than for DNA; ADP-ribose polymers compete, in fact, with DNA for H1 histone binding.