The study of the structure of active center of membrane-bound cytochrome p-450 using 1-naphthoxyalcanthiols

Homologous 1-naphtoxyalcanthiols of the type 1-C10H7O(CH2)nSH (n = 2-7) are used for structural studies of the microsomal cytochrome P450 active centre. It was found that the strongest complex of thiol with P450 is formed for n = 3. Microsomal oxidation of P450 substrates aminopyrine and benz(a) pyrene is inhibited by the 1-naphtoxyalcanthiols studied. A non-monotonous dependence of pI50 on n was found, the compound with a chain length n = 3 appeared to be the most effective inhibitor. The interaction of this thiol (n = 3) with both the heme group of P450 and the hydrophobic substrate zone is supposed and the distance between these points was estimated. It is possible to employ this approach for structural studies on the active centers of different isoforms of P450.     

Distribution of Blood Types of Four Systems and AB0 Incompatibility in Migrants of a Northern Town

The distribution of blood groups of the AB0, Rh, MN, and Kell systems were studied in migrants of a northern town of reproductive age and their newborn babies. Migrants of different ethnic groups exhibited significantly different frequencies of AB0 and Rh phenotypes. Maternal sera were tested for the presence of isoimmune antibodies to determine AB0 compatibility (or incompatibility) in mother–neonate pairs. Parents with blood group combinations resulting in mother–neonate pairs incompatible for erythrocyte antigens were less likely to have offspring with the paternal AB0 phenotype.     

Disorders in the thrombocyte link of hemostasis during endotoxinemia and their correction with the inhibitor of prostaglandin biosynthesis, indomethacin

After 10 minutes of endotoxin Salmonella typhimurium (1 mg/kg) injection into rabbits thrombocytopenia appeared, the aggregation and secretory function of circulating platelets reduced, the transformation of platelet forms from disk-shaped into spheroidal took place. On the surface of plasmatic membranes the pseudopodies and aggregates examining samples of PRP were observed. Indomethacin, blockator of biosynthesis of prostaglandins results in normalisation of morphofunctional properties of platelets.     

RET and NRG1 interplay in Hirschsprung disease

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex genetic disorder of the enteric nervous system (ENS) characterized by the absence of enteric neurons along a variable length of the intestine. While rare variants (RVs) in the coding sequence (CDS) of several genes involved in ENS development lead to disease, the association of common variants (CVs) with HSCR has only been reported for RET (the major HSCR gene) and NRG1. Importantly, RVs in the CDS of these two genes are also associated with the disorder. To assess independent and joint effects between the different types of RET and NRG1 variants identified in HSCR patients, we used 254 Chinese sporadic HSCR patients and 143 ethnically matched controls for whom the RET and/or NRG1 variants genotypes (rare and common) were available. Four genetic risk factors were defined and interaction effects were modeled using conditional logistic regression analyses and pair-wise Kendall correlations. Our analysis revealed a joint effect of RET CVs with RET RVs, NRG1 CVs or NRG1 RVs. To assess whether the genetic interaction translated into functional interaction, mouse neural crest cells (NCCs; enteric neuron precursors) isolated from embryonic guts were treated with NRG1 (ErbB2 ligand) or/and GDNF (Ret ligand) and monitored during the subsequent neural differentiation process. Nrg1 inhibited the Gdnf-induced neuronal differentiation and Gdnf negatively regulated Nrg1-signaling by down-regulating the expression of its receptor, ErbB2. This preliminary data suggest that the balance neurogenesis/gliogenesis is critical for ENS development.     

Cloning the gene for vaccinia virus strain L-IVP growth factor in Escherichia coli]

The growth factor gene of the vaccinia virus LIVP strain has been primarily cloned in a 4.3 kbp long BamHI-EcoRI fragment and then subcloned in a 440 bp fragment. It was shown that clone 4 of the LIVP strain contains a single copy of this gene while the WR strain contains a repeat. The gene is located on a 4.3 kbp BamHI-EcoRI fragment but not on a 2.2 kbp fragment and has four nucleotide changes, three of which result in amino acid substitutions.     

Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis

Ophiobolin A, a fungal toxin that affects maize and rice, has previously been shown to inhibit calmodulin by reacting with the lysine (Lys) residues in the calmodulin. In the present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin molecule by site-directed mutagenesis, either by deleting them or by changing them to glutamine or arginine. We found that each of these three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding site and is responsible for all of the inhibition of ophiobolin A. When Lys-75 was removed, Lys-77 could then react with ophiobolin A to produce inhibition. Lys-148 was shown to be a binding site but not an inhibition site. The Lys-75 mutants were partially resistant to ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues were mutated, the resulting calmodulins were very resistant to ophiobolin A. Furthermore, Lys residues added in positions 86 and/or 143 (which are highly conserved in plant calmodulins) did not react with ophiobolin A. None of the mutations seemed to affect the properties of calmodulin. These results show that ophiobolin A reacts quite specifically with calmodulin.     

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)₂ complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease.     

Acteoside improves survival in cecal ligation and puncture-induced septic mice via blocking of high mobility group box 1 release

Acteoside, an active phenylethanoid glycoside, has been used traditionally as an anti-inflammatory agent. The molecular mechanism by which acteoside reduces inflammation was investigated in lipopolysaccharide (LPS)-induced Raw264.7 cells and in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. In vitro, acteoside inhibits high mobility group box 1 (HMGB1) release and iNOS/NO production and induces heme oxygenase-1 (HO-1) expression in a concentration-dependent manner, while HO-1 siRNA antagonizes the inhibition of HMGB1 and NO. The effect of acteoside is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and Nfr2 siRNA, indicating that acteoside induces HO-1 via p38 MAPK and NF-E2-related factor 2 (Nrf2). In vivo, acteoside increases survival and decreases serum and lung HMGB1 levels in CLP-induced sepsis. Overall, these results that acteoside reduces HMGB1 release and may be beneficial for the treatment of sepsis.     

Declining carbohydrate content of Sitka-spruce treesdying from seawater exposure

Increasing sea levels associated with climate change threaten the survival of coastal forests, yet the mechanisms by which seawater exposure causes tree death remain poorly understood. Despite the potentially crucial role of nonstructural carbohydrate (NSC) reserves in tree survival, their dynamics in the process of death under seawater exposure are unknown. Here we monitored progressive tree mortality and associated NSC storage in Sitka-spruce (Picea sitchensis) trees dying under ecosystem-scale increases in seawater exposure in western Washington, USA. All trees exposed to seawater, because of monthly tidal intrusion, experienced declining crown foliage during the sampling period, and individuals with a lower percentage of live foliated crown (PLFC) died faster. Tree PLFC was strongly correlated with subsurface salinity and needle ion contents. Total NSC concentrations in trees declined remarkably with crown decline, and reached extremely low levels at tree death (2.4% and 1.6% in leaves and branches, respectively, and 0.4% in stems and roots). Starch in all tissues was almost completely consumed, while sugars remained at a homeostatic level in foliage. The decreasing NSC with closer proximity to death and near zero starch at death are evidences that carbon starvation occurred during Sitka-spruce mortality during seawater exposure. Our results highlight the importance of carbon storage as an indicator of tree mortality risks under seawater exposure.

Decline in carbohydrate storage is strongly associated with progressive mortality of trees under frequent seawater exposure, and starch is almost completely consumed at crown death.     

Brain monoamines and the hypophyseo-gonadal system of adult rats after the androgenization of their mothers during pregnancy

The brain noradrenaline system and its involvement in the control of the hypothalamic-hypophysial-gonadal complex were studied on 3 month old Wistar rats, which descended from the mothers treated with testosterone propionate on the 16th and 18th day of pregnancy. A season-independent weakening of the activating effect of noradrenaline, introduced into the brain lateral ventricle on the estradiol and testosterone content of the peripheral blood has been shown, compared with the descendants of the mothers treated with oil at the same times of pregnancy. Unlike noradrenaline, the intraventricular introduction of serotonin or dopamine was accompanied by a similar activation of the hypophysial-gonadal system in the descendants from both the androgenized and control mothers. In the descendants from the former, the thermal reaction to the intraventricular introduction of noradrenaline underwent changes as well. A conclusion is reached that testosterone propionate, introduced during the prenatal period, exerts a long-term and to a certain extent, selective and generalized effect on the developing brain noradrenaline system. In the rats, the critical period for the disturbance in androgen balance influence on the noradrenaline mechanisms of brain is timed to no later than the 18th day of prenatal development.