Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.     

Crystal structures of recombinant histones HMfA and HMfB from the hyperthermophilic archaeon Methanothermus fervidus

The hyperthermophilic archaeon Methanothermus fervidus contains two small basic proteins, HMfA (68 amino acid residues) and HMfB (69 residues) that share a common ancestry with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. HMfA and HMfB have sequences that differ at 11 locations, they have different structural stabilities, and the complexes that they form with DNA have different electrophoretic mobilities. Here, crystal structures are documented for recombinant (r) HMfA at a resolution of 1.55 A refined to a crystallographic R-value of 19.8 % (tetragonal form) and at 1.48 A refined to a R-value of 18.8 % (orthorhombic form), and for rHMfB at 1.9 A refined to a R-value of 18.0 %. The rHMfA and rHMfB monomers have structures that are just histone folds in which a long central alpha-helix (alpha2; 29 residues) is separated from shorter N-terminal (alpha1; 11 residues) and C-terminal (alpha3; 10 residues) alpha-helices by two loops (L1 and L2; both 6 residues). Within L1 and L2, three adjacent residues are in extended (beta) conformation. rHMfA and rHMfB assemble into homodimers, with the alpha2 helices anti-parallel aligned and crossing at an angle of close to 35 degrees, and with hydrogen bonds formed between the extended, parallel regions of L1 and L2 resulting in short beta-ladders. Dimerization creates a novel N-terminal structure that contains four proline residues, two from each monomer. As prolines are present at these positions in all archaeal histone sequences, this proline-tetrad structure is likely to be a common feature of all archaeal histone dimers. Almost all residues that participate in monomer-monomer interactions are conserved in HMfA and HMfB, consistent with the ability of these monomers to form both homodimers and (HMfA+HMfB) heterodimers. Differences in side-chain interactions that result from non-conservative residue differences in HMfA and HMfB are identified, and the structure of a (rHMfA)(2)-DNA complex is presented based on the structures documented here and modeled by homology to histone-DNA interactions in the eukaryal nucleosome.     

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.     

Histone HMf from the hyperthermophilic archaeon Methanothermus fervidus binds to DNA in vitro using physiological conditions

Abstract The concentration of HMf (histone Methanothermus fervidus ) in vivo has been shown to be between 1 and 2 × 10⁴ molecules per genome. At this mass ratio the amount of HMf bound to pUC19 DNA in vitro was found to be dependent on the topology of the plasmid DNA. M. fervidus grows optimally between 80 and 85°C and contains approx. 1 M K⁺ plus 300 mM 2'3'(cyclic) diphosphoglycerate. (Hensel, R. and König, H. 1988. FEMS Microbiol. Lett. 49, 75–79). HMf binding to DNA in vitro under these conditions has been demonstrated.     

DNA binding and nuclease protection by the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus

The DNA-binding and nuclease-protection properties of the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus have been shown to be consistent with the formation of nucleosome-like structures (NLS). These proteins bind to DNA molecules as short as 20 bp and form complexes that protect DNA fragments from micrococcal nuclease (MNase) digestion that are 30 bp, ∼ 60 bp and multiples of ∼ 60 bp in length. The sequences of 49 of the ∼ 60-bp DNA fragments protected from MNase digestion by HMfA have been determined and their intrinsic curvatures calculated. A circular permutation gel mobility-shift assay was used to determine directly the curvatures for five of these sequences. HMfA bound to intrinsically curved and noncurved DNAs, but exhibited a slight preference for the model curved DNA in binding competitions with a model noncurved DNA. The results obtained are consistent with the concept that the archaeal NLS is analogous, and possibly homologous, to the central core of the eukaryal nucleosome formed by a histone (H3 + H4)2 tetramer. Received: August 11, 1996 / Accepted: November 12, 1996     

Copper effect on cytochrome b of photosystem II under photoinhibitory conditions

Toxic Cu (II) effect on cytochrome b ₅₅₉ under aerobic photoinhibitory conditions was examined in two different photosystem II (PSII) membrane preparations active in oxygen evolution. The preparations differ in the content of cytochrome b ₅₅₉ redox potential forms. Difference absorption spectra showed that the presence of Cu (II) induced the oxidation of the high-potential form of cytochrome b ₅₅₉ in the dark. Addition of hydroquinone reduced the total oxidized high-potential form of cytochrome b ₅₅₉ present in Cu (II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of cytochrome b ₅₅₉ during photoinhibitory treatment showed slower kinetics of Cu (II) effect on cytochrome b ₅₅₉ in comparison with the rapid loss of oxygen evolution activity in the same conditions. This result indicates that cytochrome b ₅₅₉ is affected after PSII centres are photoinhibited. The high-potential form was more sensitive to toxic Cu (II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of cytochrome b ₅₅₉ was completely lost; however, the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. The results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of cytochrome b ₅₅₉, respectively.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation

Borrelia hermsii , a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro . Platelet binding was promoted by the platelet integrin α_IIbβ₃: the bacterium bound to purified integrin α_IIbβ₃, and bacterial binding to platelets was diminished by α_IIbβ₃ antagonists or by a genetic defect in this integrin. Integrin α_IIbβ₃ undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of α_IIbβ₃ that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin α_IIbβ₃ activation, as indicated by the expression of the platelet activation marker P-selectin and integrin α_IIbβ₃ in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I₂, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.     

Occurrence of aminopropylcadaverine and its aminopropyl derivatives aminopentylnorspermidine and N, N' -bis(3-aminopropyl)cadaverine in Halococcus acetoinfaciens

Abstract Besides putrescine, cadaverine, spermidine, spermine and thermospermine, three novel polyamines were detected in a slightly halophilic eubacterium Halococcus acetoinfaciens (IAM 12094, ATCC 25861). These novel polyamines were found to be N -3-aminopropylcadaverine [NH₂(CH₂)₃NH(CH₂)₅NH₂] and its aminopropyl derivatives: aminopentylnorspermidine [NH₂(CH₂)₃NH(CH₂)₃NH(CH₂)₅NH₂] and N , N ' -bis(3-aminoprophyl)cadaverine [NH₂(CH₂)₃ NH(CH₂)₅NH(CH₂)₃NH₂]. Aminopropylcadaverine was also detected in two other species, Halococcus agglomeratus (IAM 12095, ATCC 25862) and Halococcus nondenitrificans (IAM 12096, ATCC 25863).     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Three parameters comprehensively describe the temperature response of respiratory oxygen reduction

Using an exponential model that relies on Arrhenius kinetics, we explored Type I, Type II and dynamic (e.g. declining Q ₁₀ with increasing temperature) responses of respiration to temperature. Our Arrhenius model provides three parameters: R _REF (the base of the exponential model, nmol g⁻¹ s⁻¹), E ₀ (the overall activation energy of oxygen reduction that dominates its temperature sensitivity, kJ mol⁻¹) and δ (that describes dynamic responses of E ₀ to measurement temperature, 10³ K²). Two parameters, E ₀ and δ , are tightly linked. Increases in overall activation energy at a reference temperature were inversely related to changes in δ . At an E ₀ of ca. 45 kJ mol⁻¹, δ approached zero, and respiratory temperature response was strictly Arrhenius-like. Physiologically, these observations suggest that as contributions of AOX to combined oxygen reduction increase, E ₀( REF ) decreases because of different temperature sensitivities for V _max, and δ increases because of different temperature sensitivities for K _1/2 of AOX and COX. The balance between COX and AOX activity helps regulate plant metabolism by adjusting the demand for ATP to that for reducing power and carbon skeleton intermediates. Our approach enables determination of respiratory capacity in vivo and opens a path to development of process-based models of plant respiration.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Coat colour inheritance in Soay, Orkney and Shetland sheep

Analysis of the numerical proportions of Soay, Orkney and Shetland sheep of different colours together with test matings, produced results compatible with the hypothesis that these breeds have a multiple allelic series at locus A , white ( A ₁) being dominant to grey ( A ₂) and both being dominant to the gene for self-colour ( A ₅). The alleles at the A locus are epistatic to the alleles for pigment production at locus B , black ( B ₁) being dominant to brown ( B ₂).     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

In Vitro and In Vivo Expression of Opioid and σ Receptors in Rat C6 Glioma and Mouse N18TG2 Neuroblastoma Cells

Abstract: Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for δ opioid binding using [³H][ d -Ala², d -Leu⁵]enkephalin and for σ₁ and σ₂ binding with 1,3-[³H]di- o -tolylguanidine in the presence and absence of 1 µ M pentazocine. Receptor density ( B _max) and affinity ( K _D) were estimated by homologous competition binding assays. Opioid and σ B _max values in the solid tumors were significantly lower than their original levels in vitro. K _D values for opioid/σ ligands were similar in vitro and in vivo. With successive passages in the murine host, δ opioid and σ₁ binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, σ₂ receptor B _max values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of δ opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for σ binding was more complex, with the σ₂ response in late passages of the glioma being reminiscent of the formerly observed increase in number of σ sites in transformed human meninges, kidney, and colon tissue.     

Effect of rising temperature on the heat resistance of Listeria monocytogenes in meat emulsion

The heat resistance of a strain of L. monocytogenes was determined both in broth and in meat emulsion. The D -values for meat emulsion were approximately two to three times higher than those for broth and also the z -value increased significantly. The micro-organism proved to be more resistant when the cells were heated up slowly (0·5°C/min) to constant temperatures of 60, 63 and 66°C in meat emulsion. The D ₆₀, D ₆₃ and D ₆₆ were, respectively 12·95, 5·4 and 2·3 min. Results may have implications in the survival of Listeria monocytogenes in particular food preparations.