Conformational features of 2′-O-methyl-adenosylyl-adenosine

In order to obtain information about the conformational features of a 2′-O-methylated polyribonucleotide at the nearest neighbor level, a detailed nuclear magnetic resonance study of AmpA was undertaken. AmpA was isolated from alkali hydrolysates of yeast RNA, and proton spectra were recorded at 100 MHz in the Fourier transform mode in D₂O solutions, 0.01 M, pH 5.4 and 1.5 at 25°C. ³¹P spectra were recorded at 40.48 MHz. Complete, accurate sets of nmr parameters derived for each nucleotidyl unit by simulation iteration methods. The nmr data were translated into conformational parameters for all the bonds using procedures developed in earlier studies from these laboratories. It is shown that AmpA exists in aqueous solution with a flexible molecular framework, which shows preferences for certain orientations. The ribose rings exist as a ²E ? ³E equilibrium with the —pA ribose showing a bias for the ³E pucker. The C(4′)—C(5′) bonds of both nucleotidyl units show significant preference (75–80%) to exist in gg conformation. The dominant conformer (80%) about C(5′)—O(5′) of the 5′-nucleotidyl unit is g′g′. Even though an unambiguous determination of the orientation of the 3′-phosphate group cannot be made, tentative evidence shows that it preferentially occupies g⁺ domains [O(3′)—P trans to C(3′)—C(2′)] in which the H(3′) —C(3′)—O(3′)—P(3′) dihedral angle is about 31°. There is reasonable evidence that the 2′-O-methyl preferentially occupies the domain in which the O(2′)—CH₃ bond is trans to C(2′)—C(1′). Lowering of pH to 1.5, which results in protonation of both the adenine moieties, causes destacking of AmpA. Such destacking is accompanied by small, but real, perturbations in the conformations about most of the bonds in the backbone. A detailed comparison of the solution conformations of ApA and AmpA clearly shows that 2′-O-methylation strongly influences the conformational preference about the C(3′)—O(3′) bond of the 3′-nucleotidyl unit, in addition to inducing small changes in the overall ribophosphate backbone conformational equilibria. The effect of 2′-O-methylation is such that the C(3′)—O(3′) is forced to occupy preferentially the g⁺ domain rather than the normally preferred g^? domain [O(3′)—P trans to C(3′)—C(4′)] in ApA. The data on ApA and AmpA further reveal that the extent of stacking interaction is less in AmpA compared to ApA. It is suggested that stacked species of AmpA exist as right-handed stacks where the magnitude of ω and ω′ about O(5′)—P and P—O(3′) is about 290°. The reason for the lesser degree of stacking in AmpA compared to ApA is intramolecular interaction between 2′-O-methyl and the flexible O(3′)—P—O(5′) bridge, the interaction causing some perturbation in the magnitudes of ω/ω′, causing destacking. The destacking will lead to an increase in _χCN by a few degrees, causing an increase in ²E populations; the latter in turn will shift the 3′ phosphate group from g^? to g⁺ domains. In short, a coupled series of conformational events is envisioned at the onset of destacking, made feasible by the interaction between the 2′-O-methyl group and the swivel O(3′)—P—O(5′) bridge.     

The immunoelectrophoretic assay of insulin-I131 interaction with human α2-macroglobulinin vitro

Взаимодействие инсулина-J¹³¹ с сывороткой нормальных и лечившихся инсулином людей изучалось in vitro с помощью радиоиммуноэлектрофореза. Авторадиография, проведенная на тех же пластинках, обнаружила значительное повышение радиоактивности α₂-Макроглобулином преципитата после инкубации смеси инсулина с сывороткой при 37° C по сравнению с инкубацией этой смеси при 4° C. Эта активность наблюдалась как в нормальньх, так и в диабетических сыворотках, однако в случае диабетической сыворотки—только в избытке меченого инсулина и в завиаимости от титра антител.—Остается открытым вопрос, вызывается ли вышеописанное связывание взаимодействием α₂-Макроглобулинома с нативным инсулином—или с продуктами его деградации.     

Dynein intermediate chain 2c (DNCI2c) complex is essential for exiting Mad2-dependent spindle assembly checkpoint

The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2–p31^comet complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis.Herein, we showed that dynein intermediate chain 2c (DNCI2c)—a subunit of dynein motor protein—forms an immunocomplex with p31^comet during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31^comet overexpression. However, the combination of p31^comet overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c–p31^comet complex plays an important role in exiting Mad2-dependent SAC.     

Association between single nucleotide polymorphisms of TPH1 and TPH2 genes,and depressive disorders

Tryptophan catabolites pathway disorders are observed in patients with depression. Moreover, single nucleotide polymorphisms of tryptophan hydroxylase genes may modulate the risk of depression occurrence. The objective of our study was to confirm the association between the presence of polymorphic variants of TPH1 and TPH2 genes, and the development of depressive disorders. Six polymorphisms were selected: c.804‐7C>A (rs10488682), c.‐1668T>A (rs623580), c.803+221C>A (rs1800532), c.‐173A>T (rs1799913)—TPH1, c.‐1449C>A (rs7963803), and c.‐844G>T (rs4570625)—TPH2. A total of 510 DNA samples (230 controls and 280 patients) were genotyped using TaqMan probes. Among the studied polymoorphisms, the G/G genotype and G allele of c.804‐7C>A—TPH1, the T/T homozygote of c.803+221C>A—TPH1, the A/A genotype and A allele of c.1668T>A—TPH1, the G/G homozygote and G allele of c.‐844G>T—TPH2, and the C/A heterozygote and A allele of c.‐1449C>A—TPH2 were associated with the occurrence of depression. However, the T/T homozygote of c.‐1668T>A—TPH1, the G/T heterozygote and T allele of c.‐844G>T—TPH2, and the C/C homozygote and C allele of c.‐1449C>A—TPH2 decreased the risk of development of depressive disorders . Each of the studied polymorphisms modulated the risk of depression for selected genotypes and alleles. These results support the hypothesis regarding the involvement of the pathway in the pathogenesis of depression.     

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system

The distribution of the P2X₂ subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X₂ receptors was localised in: (i) paraventricular and supraoptic nuclei—in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis—in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X₂-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X₂-positive axons on P2X₂-positive endocrine cell bodies as well as on P2X₂-negative somata were also observed. The functional significance of these findings is discussed.     

Bacteriolytic activity of human interleukin-2

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme — chicken egg white lysozyme — by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.     

New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety—a surface recognition domain introduced to increase in cellular uptake—and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45 mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.     

Differential Effects of the Alkylating Agent N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline on Brain α2-Adrenoceptors and I2-Imidazoline Sites In Vitro and In Vivo

Abstract— The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly α₂-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l₂-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10^?8-10^?5M) markedly decreased (20–90%) the specific binding of the selective antagonist [³H]R821002 to α₂-adrenoceptors without affecting that of [³H]idazoxan (in the presence of adrenaline) to l₂-imidazoline sites. In EEDQ-pretreated membranes (10^?5M, 30 min at 25°c), the density of l₂-imidazoline sites (B_max= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10^?6M (-)-adrenaline (B_max= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [³H]idazoxan binding sites (B_max= 107 ± 6 fmol/mg of protein) (l₂-imidazoline sites plus a₂-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [³H]-R821002 to α₂-adrenoceptors, but also the binding of [³H]idazoxan to l₂-imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced α₂-adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ-induced inactivation of l₂-imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase-A sites labeled by [³H]Ro 41–1049 or that of monoamine oxidase-B sites labeled by [³H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [³H]idazoxan to l₂-imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ-pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l₂-imidazoline sites when using [³H]-imidazoline ligands that also recognize α₂-adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l₂-imidazoline sites probably by an indirect mechanism. Key Words: Brain l₂-imidazoline sites—[³H]-Idazoxan—α₂-Adrenoceptors—[³H] R821002—N -Ethoxycarbonyl-2-ethoxy-li2-dihydroquinoline—Monoamine oxidase-A—[³H]Ro 41–1049—Monoamine oxidase-B—[³H]Ro 19–6327.     

Isolation,characterization and transposition of an (IS2)2 intermediate

We have isolated and characterized a dimer derivative of the extensively studiedEscherichia coli insertion sequence IS2. The dimer structure — called (IS2)₂ — consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)₂ dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)₂ into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)₂ dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)₂ indicates that this is an active transposition intermediate. The mode of transposition of (IS2)₂ is analogous to the joined dimer model described in the case of (IS21)₂ and (IS30)₂.     

First Globus-M2 Results

Plasma Physics Reports - Globus-M2—a new 1-Tesla spherical tokamak—was recently launched. The main features and research directions of this machine in scope of fusion–fission...     

Greenhouse gas budget of a poplar bioenergy plantation in Belgium: CO2 uptake outweighs CH4 and N2O emissions

Biomass from short‐rotation coppice (SRC) of woody perennials is being increasingly used as a bioenergy source to replace fossil fuels, but accurate assessments of the long‐term greenhouse gas (GHG) balance of SRC are lacking. To evaluate its mitigation potential, we monitored the GHG balance of a poplar (Populus) SRC in Flanders, Belgium, over 7 years comprising three rotations (i.e., two 2 year rotations and one 3 year rotation). In the beginning—that is, during the establishment year and during each year immediately following coppicing—the SRC plantation was a net source of GHGs. Later on—that is, during each second or third year after coppicing—the site shifted to a net sink. From the sixth year onward, there was a net cumulative GHG uptake reaching ?35.8 Mg CO₂ eq/ha during the seventh year. Over the three rotations, the total CO₂ uptake was ?51.2 Mg CO₂/ha, while the emissions of CH₄ and N₂O amounted to 8.9 and 6.5 Mg CO₂ eq/ha, respectively. As the site was non‐fertilized, non‐irrigated, and only occasionally flooded, CO₂ fluxes dominated the GHG budget. Soil disturbance after land conversion and after coppicing were the main drivers for CO₂ losses. One single N₂O pulse shortly after SRC establishment contributed significantly to the N₂O release. The results prove the potential of SRC biomass plantations to reduce GHG emissions and demonstrate that, for the poplar plantation under study, the high CO₂ uptake outweighs the emissions of non‐CO₂ greenhouse gases.     

Paraplacental passage of 3H-prostaglandin F2

Prostaglandins F_2α permeates the live and denaturated whole fetal membranes as well as their isolated components chorion and amnion. The permeability constant in our vitro method is 1.3 × 10^?3 cm/min for PG. If 40 mg PG are instilled intra-amniotically at term — as used in therapeutical abortion — then 3.7 mg prostaglandin F_2α would pass per hour from the amniotic fluid through the fetal membranes into the decidua.     

Effect of Siberian tree species on N2O production and consumption

The effect of six Siberian tree species on two stages of denitrification—N₂O production and consumption—was studied. Broadleaf species (aspen and birch) proved to have lower rates of N₂O consumption compared to coniferous species. The factors influencing production and consumption of N₂O were also evaluated. The replacement of coniferous forests with broadleaf trees will double the N₂O/N₂ ratio in the denitrification end-products. Doubled N₂O emission from Siberian forest soils to the atmosphere can be expected due to changes in tree species composition of forest ecosystems even without considering changes in water and temperature regimes in soil.     

Cu2+-hyaluronic acid complex: Spectrophotometric detection

Hyaluronic forms a complex with Cu²⁺ showing an absorption band at 239 nm. The results indicate a 2:1 polymer-Cu²⁺ ratio in the complex formation with an equilibrium constant, 3 × 10³. Glucuronic acid, one of the monomers of hyaluronic acid, reacts with the cupric ion, showing a similar band at 235 nm, but the complex formation involves multiple equilibria. No complex formation was detected with N-acetylglucosamine—the other monomer of hyaluronic acid—and Cu²⁺. The absorption bands of the copper complexes with the polymer and glucuronic acid are attributed to a charge-transfer involving ligand to the metal ion.     

DL-α-白氨酸与稀土硝酸盐固体配合物的合成及性质

用白氨酸分别和三种稀土硝酸盐在有机溶剂中首次合成了三种相应的1:2型固体配合物,经化学分析和元素分析确定了其组成为RE(Leu)_2(NO_3)_3·2H_2O(RE=Ce,Nd,Sm;Leu=DL-α-Leucine)。并通过X—粉晶衍射、红外光谱、TG—DTG和电导等对其性质进行了讨论。     

Phenotype frequencies of vitamin D binding protein (Gc) and of posttransferrin-2 (Ptf-2) in Belgian cattle breeds*

The vitamin D binding protein (Gc) and posttransferrin-2 (Ptf-2) phenotypes have been determined in a number of Belgian cattle breeds. A very slow migrating variant of the Gc protein — Gc C — has been found in White and Red East Flemish breed. This variant was absent from the other breeds studied. This slow variant was identified as a vitamin D binding protein by autoradiography. The Gc C protein was shown to be controlled by a codominant autosomal allele Gc C at the Gclocus. The Gc C protein is probably identical with a fraction previously described in buffalo and an Italian cattle breed. The allele frequencies for the Gc and Pft-2 systems are reported for several Belgian breeds of cattle.     

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography

This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)₂D₃, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)₂D and 1,25-(OH)₂D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D₃ metabolites from their vitamin D₂ analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)₂D₃ and 24(R),25-(OH)₂D₂ as well as 1,25-(OH)₂D₃ and 1,25-(OH)₂D₂.     

Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts

Background information. N‐cadherin, a member of the Ca²⁺‐dependent cell—cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N‐cadherin‐dependent cell—cell contacts in myoblasts remain unexplored. Results. In the present study, we show that N‐cadherin‐dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N‐cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N‐cadherin‐dependent cell contacts, characterized by the immobilization of a pool of N‐cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F‐actin filaments. We also demonstrated that the formation of N‐cadherin‐dependent cell—cell contacts requires a co‐ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N‐cadherin‐dependent cell—cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N‐cadherin‐dependent cell contact formation, since, via ROCK (Rho‐associated kinase) signalling and myosin 2 activation, it allows the stabilization of N‐cadherin at the cell—cell contact sites. Conclusions. We have shown that Rac1 and RhoA have opposite effects on N‐cadherin‐dependent cell—cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.     

Percentage of phagocytosis,production of O2·−, H2O2 and NO,and antioxidant enzyme activities of rat neutrophils in culture

Changes in the integrity, ultrastructure, phagocytosis capacity, and production of H₂O₂, O₂^{· −}and NO₂⁻ were evaluated in cultured neutrophils. The activities of the antioxidant enzymes (catalase—CAT, superoxide dismutase—SOD and glutathione-dependent peroxidase—GSH-Px) were measured under similar conditions. The integrity of the cells remained unchanged up to 18 h. After 24 h, the number of viable cells in culture dropped by 16 per cent. The percentage of viable cells in culture was of 72 per cent even after 72 h. An ultrastructural analysis of the cells was carried out after 3, 6, 12, 24, 48, and 72 h in culture. Neutrophils started developing morphologic changes after 24 h: decreased cell volume, abundant vacuoles (mainly around the nucleus), and also the presence of autophagic vacuoles. This period was then chosen for the study of neutrophil function and antioxidant enzyme activities. Neutrophils cultured for 24 h presented reduced phagocytosis capacity. The rates of production of H₂O₂ and O₂^{· −} remained unchanged after 24 h in culture. Concomitantly, these cells were also able to produce NO in significant amounts. The production of O₂^·− in response to PMA stimulus was lowered in 24-h cultured cells. Possibly, the production of oxygen and nitrogen reactive species accomplished with a decrease in the activities of CAT and GSH-Px play a key role for the process of apoptosis which takes place in neutrophils under these conditions. © 1998 John Wiley & Sons, Ltd.