Association between polymorphisms of eNOS and GPx-1 genes, activity of free-radical processes and in-stent restenosis

Our aim was to examine correlations between polymorphisms in five antioxidant enzymes genes, activity of free-radical processes, and the risk of restenosis after coronary artery stenting with bare metal stents (BMS). A total of 101 male patients who underwent intracoronary stenting using BMS and coronary angiography follow-up of 6 months were enrolled in: group with in-stent restenosis (n = 44) and without restenosis (n = 57). The content of lipoperoxides and malondialdehyde (MDA) in Low-density lipoprotein (LDL), activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) in erythrocytes, and genotypes polymorphisms of the CAT gene (?262C/T), paraoxonase-1 (PON-1) gene (163T/A and 575A/G), endothelial nitric oxide synthase (eNOS) gene (298G/T (rs#1799983) and ?786T/C), GPx-1 gene (599C/T (rs#1050450)), and glutathione-S-transferase (GSTP) gene (313A/G) were determined. In carriers of the minor allele of 599C/T polymorphism of the GPx-1 gene, activity of GPx in erythrocytes was lower by 17 % than in wild allele homozygotes, while the content of lipoperoxides in LDL was higher by 74 %. T-allele of 599C/T polymorphism of the GPx-1 gene (OR = 2.9; 95 % CI: 1.23–6.82) and T-allele of 298G/T polymorphism of the eNOS gene (OR = 2.79; 95 % CI: 1.17–6.66) were associated with the risk of in-stent restenosis. Minor alleles of polymorphisms 298G/T of the eNOS gene and 599C/T of the GPx-1 gene are associated with an increased risk of in-stent restenosis. Minor allele of the GPx-1 gene 599C/T polymorphism leads to a decrease of the GPx activity and increase of the activity of free-radical processes.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Development of hybrid human interferon alfa-2 strain-producers and the use of enteropeptidase for production of N-terminal methionine-free interferons

We developed a technique for production of human interferon-α2a (IFN-α2a) and IFN-α2b lacking the N-terminal methionine. First of all, we constructed plasmids containing genes of hybrid IFN-α2 controlled by different promoters; the proximal part of the genes incorporated various sequences encoding the enteropeptidase cleavage site. As a result, four strains of Escherichia coli producing hybrid IFN-α2 were obtained. The protocol for IFN-α2 renaturation, cleavege of its N-terminal part, and chromatographic purification of the N-terminal methionine-free IFN-α2 was developed.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Structure of cephalic ganglia and their changes in the post-embryonic development of <Emphasis Type="Italic">Calliphora vomitoria</Emphasis> (L.) (Diptera,Calliphoridae)

The central nervous system of Calliphora vomitoria larvae is situated in the metathoracic and the first abdominal segments and is characterized by a high degree of oligomerization. It consists of only two ganglia: the supraoesophageal ganglion, or brain, and one large synganglion, a product of fusion of the suboesophageal ganglion, three thoracic, and all the abdominal ganglia. Weak development of the neuropil of the larval optic and olfactory lobes in the supraoesophageal ganglion is the result of a significant reduction of the head capsule and sensory organs in the larvae. The formation of the imaginal optic lobes begins at the III larval instar. The commissure of the future central body is present in the I instar larva, but formation of the imaginal structure of the central complex proceeds in the 3-day pupae and ends at the late pupal stage. The mushroom bodies are represented in the I instar larvae only by the pedunculi; the calyces can be distinguished in the II instar larvae but the final formation of their structure and the lobes of the imaginal type occurs at the pupal stage. The glomeruli in the deutocerebrum are also formed at a late stage of pupal development. Based on the degree of development of ganglia of the central nervous system, we can conclude that individual development of higher Diptera is characterized by deep de-embryonization.     

Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: structural implication for the YC-1 binding site?

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.     

Prospective register of primary percutaneous coronary interventions in patients with acute myocardial infarction

The present study has evaluated the immediate angiographic results of primary percutaneous interventions (PCI) in patients with acute myocardial infarction, as well as hospital and 6-month clinical outcomes. The analysis covered a total of 265 patients (females (23%) and males (77%)); their mean age was 57+/-11 years. The mean time before the first balloon dilatation during PCI was 278+/-135 minutes after the development of the pain syndrome or 109+/-94 minutes after hospital admission. PCI proved to be effective in 96% of the patients, as evidenced by angiography. TIMI 3 blood flow was achieved in 83% of cases during PCI. After primary PCI, hospital mortality was 98.9% and 95% survived 6 months. At 6-month follow-up, 22% patients had positive exercise tests, recurrent angina pectoris and/or more than 50% luminal stenosis of the infarct-related artery. Control angiography made less than 6 months later showed 11% restenosis. This prospective study has demonstrated the high immediate and long effectiveness and safety of primary interventions in acute myocardial infarction.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).