Clipping of Flexible Tails of Histones H3 and H4 Affects the Structure and?Dynamics of the Nucleosome

Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail’s lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA.     

Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid β

Molecular Biology - Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the β-amyloid peptide (Аβ) aggregation, which is thought to be a...     

Theoretical study of the H + HCN → H + HNC process

We present a theoretical study on the detailed mechanism and kinetics of the H+HCN →H+HNC process. The potential energy surface was calculated at the complete basis set quantum chemical method, CBS-QB3. The vibrational frequencies and geometries for four isomers (H₂CN, cis-HCNH, trans-HCNH, CNH₂), and seven saddle points (TSn where n = 1 ? 7) are very important and must be considered during the process of formation of the HNC in the reaction were calculated at the B3LYP/6-311G(2d,d,p) level, within CBS-QB3 method. Three different pathways (PW1, PW2, and PW3) were analyzed and the results from the potential energy surface calculations were used to solve the master equation. The results were employed to calculate the thermal rate constant and pathways branching ratio of the title reaction over the temperature range of 300 up to 3000 K. The rate constants for reaction H + HCN → H + HNC were fitted by the modified Arrhenius expressions. Our calculations indicate that the formation of the HNC preferentially occurs via formation of cis–HCNH, the fitted expression is k _{P W2}(T) = 9.98 × 10^?22 T ^2.41 exp(?7.62 kcal.mol^?1/R T) while the predicted overall rate constant k _{O v e r a l l} (T) = 9.45 × 10^?21 T ^2.15 exp(?8.56 kcal.mol^?1/R T) in cm ³ molecule ^?1 s ^?1.

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Graphical Abstract (a) Potential energy surface, (b) thermal rate constants as a function of temperature and (c) the branching ratios (%) of PW1, PW2, PW3 pathways involved in rm H + HCN → H + HNC process.

     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

Sequence simplicity and evolution of the 3′ untranslated region of the histone H1° Gene

The H1° gene has a long 3′ untranslated region (3′UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site. Correspondence to: P. Suau     

Colocalization of ChAT, DβH and NADPH-d in the pancreatic neurons of the newborn guinea pig

Choline acetyltransferase (ChAT) as a rate-limiting enzyme in the biosynthetic pathway of acetylcholine is thought to be present in all cholinergic neurons. However, its immunoreactivity has not been successfully applied to the study of cholinergic neurons in the pancreas. In a previous study in the pancreas of newborn guinea pig we reported the colocalization of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS) with various neuropeptides as well as dopamine-β-hydroxylase (DβH), the enzyme responsible for converting dopamine to noradrenaline. Whether NADPH-d is colocalized with ChAT in the pancreatic neurons is not known. Also it would be interesting to find out whether noradrenaline and acetylcholine could be colocalized in the same pancreatic neurons. In the present study, a method for triple labelling of ChAT, DβH and NADPH-d was used to answer the above questions. Colocalization of ChAT, DβH and NADPH-d was constantly demonstrated in the same neurons in the same sections. It is concluded that some of the pancreatic neurons may utilize more than one neurotransmitter such as nitric oxide (NO), acetylcholine and noradrenaline to achieve their function. The possible cotransmission of acetylcholine and noradrenaline was extremely intriguing, and its mechanism and significance needs to be further investigated.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Structural variability in the genome of phage ?H of Halobacterium halobium

Summary The partially circularly permuted, terminally redundant structure of the DNA of phage H has been confirmed by a cleavage map for the restriction enzymes PstI, ClaI, BglII, HindIII, and, partially, BamHI.Six variants of phage H have been isolated from 71 single plaques. Their genomes differ by several insertions, a deletion, and an inversion of a DNA segment with a minimal length of 11 kb. The inversion occurs with high frequency in variants carrying at the flanks of the invertible DNA in verted repeats of a 1.8 kb DNA element which shares sequence homology with the DNA of H. halobium and may be involved in the extreme variability of its genome.     

Proton block of the CLC-5 Cl?/H+ exchanger

CLC-5 is a H⁺/Cl⁻ exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent’s disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H⁺ transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H⁺ binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN⁻ is the main charge carrier: H⁺ transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H⁺ translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.     

Characterization of the Na⁺/H⁺ antiporter from Yersinia pestis

Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na/H antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein''s substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein''s structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na/H antiporter''s structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na/H antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.     

Hürthle cell carcinoma of the thyroid presenting as thyrotoxicosis

ObjectiveTo report a case of hyperthyroidism associated with Hülllnnvl-ürthle cellcarcinoma and to review the literature regarding this relationship.MethodsWe describe the clinical, biochemical, radiologic, and pathologic data of a patient with Hürthle cellcarcinoma associated with thyrotoxicosis and reversible heart failure. We discuss the mechanistic aspects and review previously reported cases of functionalHürthle cellcarcinomas.ResultsA 43-year-old womanpresented with thyrotoxicosis and nonischemic cardiomyopathy. She had a “hot” nodule inthe left lobe of the thyroid onsodium pertechnetate scan. She underwent a left hemithyroidec-tomy and isthmusectomy. Pathologic findings revealed a minimally invasive Hürthle cellcarcinoma. Onfollow-up, the dilated cardiomyopathy had resolved. The associationof thyroid carcinoma with thyrotoxicosis is rare.ConclusionsSome Hürthle cellcarcinomas canbe functionaland lead to thyrotoxicosis. To our knowledge, we present the first case of reversible dilated cardiomyopathy due to thyrotoxicosis originating from Hülll-ürthle cellcarcinoma. (Endocr Pract. 2012;18:e5-e9)     

Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure—a FRET study

Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.     

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G₁ cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G₂. Intriguingly, G₂ cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G₂H” compartment), to cells that there were almost negative and had about 2 foci (termed “G₂L” compartment). EdU-labeling of S phase cells revealed that G₂H was directly populated from S phase, while G₂L was populated from G₂H, but in control cells also directly from S phase. The length of G₂H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G₂H, and enter mitosis after a 2–3 h delay in G₂L.     

Effects of NR1H3 Genetic Variation on the Expression of Liver X Receptor α and the Progression of Alzheimer's Disease

Alzheimer''s disease (AD) has been postulated to involve defects in the clearance of amyloid-β (Aβ). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aβ. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aβ42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aβ42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aβ42.