H2 histaminic receptors in rat cerebral cortex. 3. Inhibition of [3H]histamine by H2 agonists

The binding of [3H]histamine to H2 receptors in homogenates of rat cerebral cortex is inhibited by 11 H2 agonists in a characteristic and unique manner. At low concentrations of the radioligand (less than 1.5 nM), the inhibitory profiles of individual agonists (A) are distinctly biphasic; specific binding is well described in most cases by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1. Maximal inhibition is the same for all agonists. Since values of F2 vary from 0.42 to 0.90, the agonist appears to determine the equilibrium distribution of receptors between two states of affinity. Ratios of apparent affinity (K2/K1) vary from 204 to 3 090 000, and there is no correlation between values of K1 and K2. Compounds lacking H2 activity, including structural analogues of histamine and dimaprit, reveal a Hill coefficient of 1 and inhibit the radioligand only weakly. For six agonists, values of K2 agree and correlate well (P = 0.00047) with H2 pharmacological potency (EC50) in the guinea pig right atrium; for the others, K2 is less than EC50 by 15-61-fold. Four observations suggest that the inhibition corresponding to F1 is allosteric and cooperative: the dissociation constant of the radioligand appears to vary in the presence of an unlabeled agonist, absolute levels of binding corresponding to F1, as defined by dimaprit, decrease at higher concentrations of [3H]histamine, F1 for dimaprit is reduced from 0.48 to 0.32 by 2-methylhistamine (F1 = 0.27) at a concentration of 20 nM (approximately K1(0.5) K2(0.5) for 2-methylhistamine), but the increase in K1 for dimprit is at least 100-fold less than expected from competitive effects, and 1 equiv of some agonists appears to preclude access of [3H]histamine to more than 1 equiv of receptors, with no evidence that an appreciable fraction of the unlabeled drug is bound. Noncompetitive effects also may account in part for the inhibition corresponding to F2.     

Nucleosome cores containing H2B,H3, H4 and HMG2 are reconstituted

Nucleosome cores mixed with the high mobility group proteins, HMG1 and HMG2, in 2 M NaCl, 5 M urea, 0.2 mM EDTA and 10 mM Tris pH 7.0, have been reconstituted by salt gradient dialysis. The reconstituted material, in 10 mM Tris pH 7.0, had a sedimentation peak at the same position as that of control nucleosome cores in sucrose density gradient ultracentrifugation. The SDS polyacrylamide gel electrophoresis of the reconstituted nucleosome cores demonstrated that they contain H2B, H3, H4 and HMG2 and are selectively deficient in H2A. The circular dichroism of DNA of the reconstituted cores was indistinguishable from that of control nucleosome cores. The results suggest that HMG2 replaces H2A as a component of the nucleosome histone core during reconstitution.     

Measurement of H2'-C2' and H3'-C3' dipolar couplings in RNA molecules

The quality of nucleic acid solution structures can be significantly improved using residual dipolar coupling data. However, many of the one-bond couplings that could be used for this purpose are difficult to measure. Conventional 2D experiments are often unable to reveal one-bond H2-C2 and H3-C3 couplings in large RNA molecules due to spectral overlap. Here we show how to use 3D HCcH-COSY and Relay HCcH-COSY to measure one-bond H2-C2 and H3-C3 couplings which improved the precision of the structures obtained recently for a 42 nucleotide RNA.     

Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.

Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.     

Structure of subnucleosomal particles. Tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes

The tetrameric (H3/H4)2 146 base pair (bp) DNA and hexameric (H3/H4)2(H2A/H2B)1 146 bp DNA subnucleosomal particles have been prepared by depletion of chicken erythrocyte core particles using 3 or 4 M urea, 250 mM sodium chloride, and a cation-exchange resin. The particles have been characterized by cross-linking and sedimentation equilibrium. The structures of the particles, particularly the tetrameric, have been studied by sedimentation velocity, low-angle neutron scattering, circular dichroism, optical melting, and nuclease digestion with DNase I, micrococcal nuclease, and exonuclease III. It is concluded that since the radius of gyration of the DNA in the tetramer particle and its maximum dimension are very close to those of the core particle, no expansion occurs on removal of all the H2A and H2B. Nuclease digestion results indicate that histones H3/H4 in the tetramer particle protect a total of 70 bp of DNA that are centrally located within the 146 bp. Within the 70 bp DNA length, the two terminal regions of 10 bp are, however, not strongly protected from digestion. The optical melting profile of both particles can be resolved into three components and is consistent with the model of histone protection of DNA proposed from nuclease digestion. The structure proposed for the tetrameric histone complex bound to DNA is that of a compact particle containing 1.75 superhelical turns of DNA, in which the H3 and H4 histone location is the same as found for the core particle in chromatin by histone/DNA cross-linking [Shick, V. V., Belyavsky, A. V., Bavykin, S. G., & Mirzabekov, A. D. (1980) J. Mol. Biol. 139, 491-517]. Optical melting of the hexamer particle shows that each (H2A/H2B)1 dimer of the core particle protects about 22 base pairs of DNA.     

Distribution of the core histones H2A.H2B.H3 and H4 during cell replication.

The distribution of newly synthesized core histones H2A, H2B, H3 and H4 relative to the DNA strand synthesized in the same generation has been examined in replicating Chinese Hamster ovary cells. Cells are grown for one generation in [14C]-lysine and thymidine, and then for one generation in [3H]-lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in unlabeled lysine and thymidine. This protocol produces equal amounts of unifilarly substituted and unsubstituted DNA. Monomer nucleosomes isolated from chromatin containing these two types of DNA can be distinguished by crosslinking with formaldehyde and banding to equilibrium in CsCl density gradients. The results indicate that the core histones are equally distributed between the two types of DNA. These findings are discussed in terms of current models for chromatin replication; they do not support any long term association of newly replicated histones with either the leading or lagging side of the replication fork.     

Regulation of H2a-specific proteolysis by the histone H3:H4 tetramer

We have studied the limited cleavage of H2a in the H2a:H2b histone dimer by the H2a-specific protease under physiological conditions (neutral pH, 0.1 M NaCl) using a variety of histone-DNA reconstitutes as substrates and/or regulators of the partially purified enzyme. Under these conditions the protease cleaves H2a in "native" dimer-DNA reconstitutes but not in "native" octamer-DNA reconstitutes. Treatment of the enzyme with saturating amounts of H3:H4 tetramer-DNA prior to addition of dimer-DNA substrate results in complete inhibition of H2a-specific proteolysis. Sucrose gradient sedimentation experiments indicate that the protease binds reversibly to tetramer-DNA and that this leads to the reversible inhibition of enzymatic activity. Using three different tetramer-DNA complexes, we found native tetramer-DNA to be a more effective inhibitor than either trypsin-treated tetramer-DNA or acetylated tetramer-DNA. We conclude that under physiological conditions, the H2a-specific protease binds primarily to the highly basic amino-terminal domain of the H3:H4 tetramer, and this binding lowers the effective concentration of enzyme available to cleave H2a. Although no cleaved H2a is produced when protease is mixed with native octamer-DNA, incubation of the enzyme with acetylated octamer-DNA results in H2a-specific proteolysis. This is the first demonstration that the H2a-specific protease activity can be modulated by a physiologically relevant process (e.g. histone acetylation). We propose that the sequestered protease may be functionally regulated in vivo through reversible post-translational modifications to the NH2-terminal domains of the histone H3:H4 tetramer.     

H2 histaminic receptors in rat cerebral cortex. 2. Inhibition of [3H]histamine by H2 antagonists

Sites labeled by [3H]histamine in homogenates of rat cerebral cortex reveal a pharmacological specificity typical of H2 receptors. Fourteen H2 antagonists inhibit the specific binding of the radioligand to the same level; Hill coefficients are near or equal to one for five compounds and markedly lower for nine. The binding patterns of individual antagonists (A) are well described by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1; F2 is 0 for those drugs that reveal a Hill coefficient of 1. Concentrations of A that reduce specific binding by 50% (IC50) correlate well (r = 0.991; P less than 0.00001) and show good numerical agreement with potencies reported for inhibition of the response to histamine in H2-mediated systems. The correlation is poorer when IC50 is replaced by either K1 (r = 0.973) or K2 (r = 0.921) for those antagonists that reveal both; the antihistaminic activity of the drug thus appears not to be associated preferentially with one or other class of sites. Since F2 varies from 0.16 to 0.60 among those antagonists that discern heterogeneity, the antagonist appears to determine the distribution of sites between the two classes. Moreover, a correlation among antagonists between values of K1 and K2 (r = 0.975; P = 0.00001) suggests that the apparent heterogeneity reflects different conformers within an otherwise homogeneous population. H2 antagonists appear to be noncompetitive with respect to each other and to the radioligand: one antagonist has relatively little effect on the values of K1, K2, and F2 revealed by another; also, estimates of K1 and K2 are independent of the concentration of [3H]histamine between 1.3 and 10 nM, although the radioligand exhibits an apparent dissociation constant of 3.9 nM [Steinberg, G. H., Eppel, J. G., Kandel, M., Kandel, S. I., & Wells, J. W. (1985) Biochemistry (preceding paper in this issue)].     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

2-Aryloxymethylmorpholine histamine H(3) antagonists

The synthesis and biological activity of a new series of 2-aryloxymethylmorpholine histamine H(3) antagonists is described. The new compounds are high affinity histamine H(3) ligands that penetrate the CNS and occupy the histamine H(3) receptor in rat brain.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

Synthesis of methyl [18-2H3], [17-2H2], [16-2H2], [14-2H2] and [12-2H2] cis-9-octadecenoates

Methyl [17-²H₂]oleate was prepared by stepwise reduction from 17-oxooleate in 24% yield. Methyl [18-²H₃], [16-²H₂], [14-²H₂] and [12-²H₂] oleates were synthesized from appropriately deuterated octylbromides by conversion to deuterated 7-hexadecyn-1-ols and chain extention to deuterated stearolates followed by semihydrogenation; overall yields were about 17%.     

Human monoclonal antibodies to pandemic 1957 H2N2 and pandemic 1968 H3N2 influenza viruses

Investigation of the human antibody response to the 1957 pandemic H2N2 influenza A virus has been largely limited to serologic studies. We generated five influenza virus hemagglutinin (HA)-reactive human monoclonal antibodies (MAbs) by hybridoma technology from the peripheral blood of healthy donors who were born between 1950 and 1968. Two MAbs reacted with the pandemic H2N2 virus, two recognized the pandemic H3N2 virus, and remarkably, one reacted with both the pandemic H2N2 and H3N2 viruses. Each of these five naturally occurring MAbs displayed hemagglutination inhibition activity, suggesting specificity for the globular head domain of influenza virus HA. When incubated with virus, MAbs 8F8, 8M2, and 2G1 each elicited H2N2 escape mutations immediately adjacent to the receptor-binding domain on the HA globular head in embryonated chicken eggs. All H2N2-specific MAbs were able to inhibit a 2006 swine H2N3 influenza virus. MAbs 8M2 and 2G1 shared the V(H)1-69 germ line gene, but these antibodies were otherwise not genetically related. Each antibody was able to protect mice in a lethal H2N2 virus challenge. Thus, even 43 years after circulation of H2N2 viruses, these subjects possessed peripheral blood B cells encoding potent inhibiting antibodies specific for a conserved region on the globular head of the pandemic H2 HA.