Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.     

A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.     

Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.     

Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.     

Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends

Protelomerases are enzymes responsible for the generation of closed hairpin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the complementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage phiKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3' end of the cleaved openings leaving a 5'-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3'-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and phiKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity.     

Influence of Interfacial Composition on in Vitro Digestibility of Emulsified Lipids: Potential Mechanism for Chitosan's Ability to Inhibit Fat Digestion

The objective of this study was to investigate the influence of interfacial composition and electrical charge on the in vitro digestion of emulsified fats by pancreatic lipase. An electrostatic layer-by-layer deposition technique was used to prepare corn oil-in-water emulsions (3 wt% oil) that contained droplets coated by (1) lecithin, (2) lecithin–chitosan, or (3) lecithin–chitosan–pectin. Pancreatic lipase (1.6 mg mL⁻¹) and/or bile extract (5.0 mg mL⁻¹) were added to each emulsion, and the particle charge, droplet aggregation, and free fatty acids released were measured. In the presence of bile extract, the amount of fatty acids released per unit amount of emulsion was much lower in the emulsions containing droplets coated by lecithin–chitosan (38 ± 16 μmol mL⁻¹) than those containing droplets coated by lecithin (250 ± 70 μmol mL⁻¹) or lecithin–chitosan–pectin (274 ± 80 μmol mL⁻¹). In addition, there was much more extensive droplet aggregation in the lecithin–chitosan emulsion than in the other two emulsions. We postulated that lipase activity was reduced in the lecithin–chitosan emulsion as a result of the formation of a relatively thick cationic layer around each droplet, as well as the formation of large flocs, which restricted the access of the pancreatic lipase to the lipids within the droplets. Our results also suggest that droplets initially coated by a lecithin–chitosan–pectin layer did not inhibit lipase activity, which may have been because the chitosan–pectin desorbed from the droplet surfaces thereby allowing the enzyme to reach the lipids; however, further work is needed to establish this. This information could be used to create food emulsions with low caloric level, or to optimize diets for individuals with lipid digestion problems.     

Peptide YY levels are elevated after gastric bypass surgery

Objective: Mechanisms that promote effective and sustained weight loss in persons who have undergone Roux‐en‐Y gastric bypass surgery are incompletely understood but may be mediated, in part, by changes in appetite. Peptide YY (PYY) is a gut‐derived hormone with anorectic properties. We sought to determine whether gastric bypass surgery alters PYY levels or response to glucose. Research Methods and Procedures: PYY and ghrelin levels after a 75‐gram oral glucose tolerance test were measured in 6 morbidly obese patients 1.5 ± 0.7 (SE) years after gastric bypass compared with 5 lean and 12 obese controls. Results: After substantial body weight loss (36.8 ± 3.6%) induced by gastric bypass, the PYY response to an oral glucose tolerance test was significantly higher than in controls (p = 0.01). PYY increased ～10‐fold after a 75‐gram glucose load to a peak of 303.0 ± 37.0 pg/mL at 30 minutes (p = 0.03) and remained significantly higher than fasting levels for all subsequent time‐points. In contrast, PYY levels in obese and lean controls increased to a peak of ～2‐fold, which was only borderline significant. Ghrelin levels decreased in a symmetric but opposite fashion to that of PYY. Discussion: Gastric bypass results in a more robust PYY response to caloric intake, which, in conjunction with decreased ghrelin levels, may contribute to the sustained efficacy of this procedure. The findings provide further evidence for a role of gut‐derived hormones in mediating appetite changes after gastric bypass and support further efforts to determine whether PYY_3–36 replacement could represent an effective therapy for obesity.     

Evaluation of toxicological monitoring markers using proteomic analysis in rats exposed to formaldehyde

Formaldehyde (FA) is known as a low molecule weight organic compound and one of major components that causes sick building syndrome (SBS), and it has been reported that FA has cytotoxic, hemotoxic, immunotoxic, and genotoxic properties. The International Agency for Research on Cancer (IARC) has characterized FA as a carcinogen. In this study, we investigated the effects of FA on rat plasma proteins by using proteomic approach. Rats were exposed to three different concentrations of FA (0, 5, 10 ppm) for 2 weeks at 6 hours/day and 5 days/week in an inhalation chamber. Malondialdehyde (MDA) assay and carbonyl spectrometric assay were conducted to determine lipid peroxidation and protein oxidation levels and Comet assays were used for genotoxicity evaluation. Level of MDA, carbonyl insertion and DNA damage in plasma, livers, and in the lymphocytes of rats exposed to FA were found to be dose dependently increased. Proteomic analysis using three different pI ranges (3.5-5.6, 5.3-6.9, 6-9) and large size two-dimensional gel electrophoresis (2-DE) showed the presence of 3491 protein spots. A total of 32 (19 up- and 13 down-regulated) proteins were identified as biomarkers of FA, all showed dose dependent expressions in the plasma of rats exposed to FA and of these, 27 protein spots were identified by MALDI-TOF/MS. Several differentiated protein groups were found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of SNAP 23, apolipoprotein A-1 and E, clusterin, kinesin, and fibrinogen gamma were confirmed by Western blot assay, and apo E was further analyzed by using 2-DE immunoblot assays to determine isoform patterns. Two cytokine including IL4 and INF-gamma were measured in plasma with respect to fibrinogen gamma changes. In summary, cytotoxicity, and genotoxicity assays, namely MDA lipid peroxidation assay, the carbonyl protein oxidation assay, and Comet genotoxic assay showed that these effects increased on increasing FA levels. Proteomic analysis with three different pI ranges and long size 2-DE gel electrophoresis showed that 32 protein spots were up-or down-regulated. Of these 32 proteins, 7 proteins were confirmed by western blot assay. They could be potential biomarkers for human diseases associated with FA exposure.     

Asian ginseng extract inhibits in vitro and in vivo growth of mouse lewis lung carcinoma via modulation of ERK‐p53 and NF‐κB signaling

Asian ginseng (AG) is the most commonly used medicinal herb in Asian countries. It is often prescribed for cancer patients as a complementary remedy. However, whether AG in fact benefits cancer patients remains unknown because some studies reported that AG facilitates tumor growth, which contradicts its usage as a dietary remedy to cancer patients. In addition, most of research works on ginseng for anti‐cancer were using single ginsenoside rather than whole root extracts used in clinics. Thus, intensive studies using the type of ginseng as its clinical form are necessary to validate its benefits to cancer patients. In this study, anti‐tumor potency and underlying molecular mechanisms of the ethanol extract of AG (EAG) were examined in mice with Lewis lung carcinoma (LLC‐1). We showed that EAG significantly suppressed tumor growth in LLC‐1‐bearing mice with concomitant down‐regulation of PCNA proliferative marker, and it exhibited specific cytotoxicity to cancer cells. EAG also induced MAPK and p53 signaling in LLC‐1 cells, which suppressed cyclin B–cdc2 complex and in turn induced G2–M arrest and apoptosis. Although EAG could activate NF‐κB signaling, the proteasome inhibitor of MG‐132 could effectively prevent NF‐κB targeted gene expression induced by EAG and then sensitize LLC‐1 cells to induce EAG‐mediated apoptosis. Collectively, EAG in a relatively high dose significantly suppressed tumor growth in LLC‐1‐bearing mice, indicating that AG may benefit lung cancer patients as a dietary supplement. This is the first report demonstrating possible combination of EAG with proteasome inhibitors could be a novel strategy in anti‐cancer treatment. J. Cell. Biochem. 111: 899–910, 2010. © 2010 Wiley‐Liss, Inc.