H19 and Igf2--enhancing the confusion?

Genomic imprinting, whereby certain genes are expressed dependent on whether they are maternally or paternally inherited, is restricted to mammals and angiosperm plants. This unusual mode of gene regulation results from the complex interplay between cis-regulatory elements, leading to parent-of-origin-dependent epigenetic modifications and tissue-specific patterns of imprinted gene expression. Many studies of imprinting and imprinted genes have focused on epigenetic effects, such as DNA methylation and chromatin structure. However, it is equally important to explore the interconnected role of regulatory elements at imprinted domains by genetic experiments, including the use of transgenes and deletions.     

γH2AX foci formation in the absence of DNA damage: Mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage.     

Do the higher oxidation states of the photosynthetic O2-evolving system contain bound H2O?

A modified mass spectrometer was used to determine whether the higher oxidation states of the photosynthetic O2-evolving system contain substrate water that is not freely exchangeable with the external medium. Our data indicated that the higher oxidation states contain no appreciable bound, non-exchangeable H2O. This suggests that H2O oxidation takes place via a rapid, concerted, all-or-none mechanism rather than by a mechanism involving stable, partially oxidized, H2O-derived intermediates. These findings set definite constraints on possible mechanisms of O2 evolution.     

Is H2 the Universal Energy Source for Long-Term Survival?

Abstract This review revisits anabiosis (cryptobiosis or latent life); but more specifically with the discrepancy (time factor) between the finding of viable bacteria in ancient material and the racemization of amino acids and depurination of DNA that would have contributed to their death. The omnipresence of H₂ in the biosphere since life began, its ability to penetrate the microbial cell, its low energy of activation, its ability to form protons and electrons in the presence of Fe(II), and its (including electrons and protons) role in many biochemical reactions make H₂ the best candidate as the energy of survival for microbial cells. Although the concentration of H₂ in most environments is below the threshold level for microbial growth, the surviving cells have a long period of time to carry out the necessary metabolism to offset the racemization and depurination processes. This paper explores a hypothesis that explains this discrepancy. Received: 30 March 1999; Accepted: 27 July 1999; Online Publication: 30 November 1999     

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.     

Relation between electron exchange and nuclear vibrations in the H 2 + +H 2 + → H 3 + +p exothermic ion-molecular reaction

The effective cross section for the H ₂ ⁺ +H ₂ ⁺ → H ₃ ⁺ +p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The maximum of the cross section at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr≈5 eV provides evidence in favor of the classical model of the H ₂ ⁺ ion with the charge fixed on one of the nuclei throughout the collision event.     

Further quasi-classical trajectory studies on the C++ H2O reaction

Further trajectory studies on the C⁺ + H₂O reaction have been performed using a potential energy surface described through a finite element method in its p version. In former trajectory studies [Y. Ishikawa, T. Ikegami and R.C. Binning Jr., Direct ab initio molecular dynamics study of C⁺+H₂O: angular distribution of products and distribution of product kinetic energies, Chem. Phys. Lett. 370 (2003), pp. 490–495; J.R. Flores, Quasichemical trajectories on a finite element density functional potential energy surface: the C⁺+H₂O reaction revisited, J. Chem. Phys. 125 (2006), 164309], tunnelling was not taken into account. The present results together with the analysis of the electronic excited states [J.R. Flores and A.B. González, The role of the excited electronic states in the C⁺+H₂O reaction, J. Chem. Phys. 128 (2008), 144310] are useful to interpret the mechanism of the title reaction, which has been the subject of crossed beam experiments [D.M. Sonnenfroh, R.A. Curtiss and J.M. Farrar, Collision complex formation in the reaction of C⁺ with H₂O, J. Chem. Phys. 83 (1985), pp. 3958–3964] and can be considered a prototypical ion–molecule reaction.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

Dependence on Mating Type for the Overproduction of Iso-2-Cytochrome c in the Yeast Mutant CYC7–H2

The CYC7–H2 mutation causes an approximately 20-fold overproduction of iso–2–cytochromo c in a and α haploid strains of the yeast Saccharomyces cerevisiae due to an alteration in the nontranslated regulatory region that is presumably contiguous with the structural region. In this investigation, we demonstrated that heterozygosity at the mating type locus, a/α or a/a/α/α, prevents expression of the overproduction, while homozygosity, a/a and α/α, and hemizygosity, a/0 and α/0, allow full expression of the CYC7–H2 mutation, equivalent to the expression observed in a and α haploid strains. There is no decrease in the overproduction of iso-2-cytochrome c in a/α diploid strains containing either of the other two similar mutations, CYC7–H1 and CYC7–H3. It appears as if active expression of one or another of the mating-type alleles is required for the overproduction of iso-2-cytochrome c in CYC7–H2 mutants.     

Vacuolar H+/Ca2+ transport: who's directing the traffic?

Physiological studies have established the role of plant high-capacity vacuolar H+/Ca2+ exchange activity in ion homeostasis and signal transduction. The molecular characterization and structure-function analyses of these transporters are just beginning to emerge. In yeast, Ca2+ signaling molecules regulate vacuolar H+/Ca2+ exchange. Recently, some of the Ca2+ dependent "molecular relay" molecules have been characterized in plants; however, the regulation of plant vacuolar H+/Ca2+ exchange remains an open question.     

Estradiol -17-β enhances the formation of {3H}-PGF2α from {3H}-PGE2 in the uterus isolated from ovariectomized rats

Formation of {³H}-PGF_2α and {³H}-13,14,dihydro-15-keto-PGF_2α from {³H}-PGE₂ by the supernatant of uterine homogenates from estrous and ovariectomized rats, was studied, using the reaction system PGE₂ + NADPH + {³H}-PGE₂ + supernatant. Enzymatic conversion was lower in uterine supernatants from spayed rats than in uterine homogenates of rats at natural estrus.Spayed animals were injected with progesterone (P) or with estradiol-17-β (E₀) at a dose of 1.0 or 50.0 ug. Conversion of {³H}-PGF_2α to {³H}-PGE₂ or to {³H}-13,14,dihydro-15-keto-PGF_2α did not differ in control ovariectomized or ovariectomized rats receiving P or 1.0 ug E₀. However, 50.0 ug E₀ induced a significant oversion after 30 (P < 0.01) and 60 (P < 0.001) min of incubation.It is concluded that E₀, at the 50.0 ug dose, but not the 1.0 ug dose of E₀, nor progesterone, stimulated conversion of {³H}-PGE₂ into {³H}-PGF_2α or {³H}-13, 14,dihydro-15-keto-PGF_2α, presumably through the activity of the enzyme PGE₂-9-keto-reductase.     

How relevant are S=O and P=O Double Bonds for the Description of the Acid Molecules H2SO3, H2SO4, and H3PO4, respectively?

The acid molecules H₂SO₃, H₂SO₄, and H₃PO₄ are usually drawn using "Lewis structures" which exhibit the octet extension by 3d-orbitals on sulfur and phosphorus, respectively. Thus, S=O and P=O double bonds are assumed to be formed. The natural d-orbital occupancies on S and P, however, were calculated to be as low as 0.1 e, and therefore, an octet extension can hardly be expected. After the natural bond orbitals (NBO) search procedure was forced to attempt to form different Lewis structures of bonds and lone pairs, we defined the optimal Lewis structure, if a dominant structure exists at all, by the maximum electronic charge in Lewis orbitals. Indeed, sulfur obeys the octet rule in the optimal zwitterionic Lewis structures and does not form S=O double bonds. No dominant resonance structure could be found for H₃PO₄ where polarized PO ?-bond and zwitterionic PO bond structures exhibit similar weights.     

Does the rat have an H2-D orthologue next to Bat1?

Unlike all other mammalian species, which have only one class I region, rat and mouse possess a second class I region on the centromeric side of the MHC. The mouse has class Ia genes in both the centromeric H2-K and the telomeric H2-D region, whereas the rat has class Ia genes only in the centromeric RT1.A region. Bat1 is the last gene of the class III region in the mouse, and H2-D was mapped 10 kb telomeric of Bat1. To determine whether the rat possesses an H2-D orthologue, we sequenced a cosmid clone that contains rat Bat1 and an adjacent class I gene, RT1.46 (l). Homology searches suggest a transition in the rat sequence with a proximal stretch containing Nfkbil1, ATP6G, and Bat1, which is homologous to the mouse H2-D region, and a more-distal stretch, which contains the class I gene and has many similarities to mouse H2-Q region sequences. Downstream of Bat1 is a sequence stretch with great similarity to intron 3 of H2-D, which is not present in any of the rat class I genes but is found in mouse H2-K, D-, and - Q region genes. Numerous repetitive elements indicate that the region is prone to repeat-mediated rearrangements. A putative H2-D orthologue may have been present at this location and lost by genomic rearrangements, leaving the short intronic sequence behind. The class I gene RT1.46 (l) has an open reading frame, but it is unlike H2-D due to a unique 5'UTR shared with H2-Q1 and Q2, the absence of the B2 SINE repeat characteristic of H2-D/L, and the apparent lack of surface expression. We conclude that at least the LEW rat (RT1 (l)) does not possess an H2-D orthologue.     

Molecular basis of the apolipoprotein H (β2-glycoprotein I) protein polymorphism

Apolipoprotein H (apoH, protein; APOH, gene) is considered to be an essential cofactor for the binding of certain antiphospholipid autoantibodies to anionic phospholipids. APOH exhibits a genetically determined structural polymorphism due to the presence of three common alleles (APOH*1, APOH*2 and APOH*3 ) detectable by isoelectric focusing (IEF) and immunoblotting. The APOH*3 allele can be further characterized into two subtypes, APOH*3^W and APOH*3^B, based upon its reactivity with monoclonal antibody 3D11. In this study we have determined the molecular basis of the APOH protein polymorphism and its distribution in three large U.S. population samples comprising 661 non-Hispanic whites, 444 Hispanics and 422 blacks. By direct DNA sequencing of PCR amplified fragments corresponding to the eight APOH exons, we identified two missense mutations that correspond to the APOH*1 and APOH*3^W alleles. A missense mutation (G→A) in exon 3, which alters amino acid Ser to Asn at codon 88 and creates a restriction site for TSP509 I, was present in all APOH*1 allele carriers. A second missense mutation (G→C) at codon 316 in exon 8, which replaces amino acid Trp with Ser and creates a restriction site for BSTBI, was present in all APOH*3^W carriers. The distribution of the Ser 88 Asn and Trp 316 Ser mutations was significantly different between the three racial groups. The frequency of the Asn-88 allele was 0.011, 0.043, and 0.056 in blacks, Hispanics and non-Hispanic whites, respectively. While the Ser-316 allele was observed sporadically in blacks (0.008), it was present at a polymorphic frequency in Hispanics (0.027) and non-Hispanic whites (0.059). The identification of the molecular basis of the APOH protein polymorphism will help to elucidate the structural – functional relationship of apoH in the production of antiphospholipid autoantibodies. Received: 20 November 1996 / Accepted: 13 February 1997     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

Quantitative and time-course analysis of microbial degradation of 1H,1H,2H,2H,8H,8H–perfluorododecanol in activated sludge

A methylene group in the fluorinated carbon backbone of 1H,1H,2H,2H,8H,8H–perfluorododecanol (degradable telomer fluoroalcohol, DTFA) renders the molecule cleavable by microbial degradation into two fluorinated carboxylic acids. Several biodegradation products of DTFA are known, but their rates of conversion and fates in the environment have not been determined. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitatively investigate DTFA biodegradation by the microbial community in activated sludge in polyethylene terephthalate (PET) flasks, which we also determined here showed least adsorption of DTFA. A reduction in DTFA concentration in the medium was accompanied by rapid increases in the concentrations of 2H,2H,8H,8H–perfluorododecanoic acid (2H,2H,8H,8H–PFDoA), 2H,8H,8H-2-perfluorododecenoic acid (2H,8H,8H-2-PFUDoA), and 2H,2H,8H-7-perfluorododecenoic acid and 2H,2H,8H-8-perfluorododecenoic acid (2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA), which were in turn followed by an increase in 6H,6H–perfluorodecanoic acid (6H,6H–PFDeA) concentration, and decreases in 2H,2H,8H,8H–PFDoA, 2H,8H,8H-2-PFUDoA, and 2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA concentrations. Accumulation of perfluorobutanoic acid (PFBA), a presumed end product of DTFA degradation, was also detected. Our quantitative and time-course study of the concentrations of these compounds reveals main routes of DTFA biodegradation, and the presence of new biodegradation pathways.