Synthesis of guanidino analogues of PMPDAP and their immunobiological activity

A series of novel 9-, 7- and 3-substituted 2- or 6-guanidinopurines as analogues of potent antiviral and immunobiologically active compound enantiomers of PMPDAP was synthesized and evaluated for their biological activity. Compounds containing the combination of guanidino and amino group at the purine moiety enhanced the interferon-gamma-triggered NO production in murine macrophages and stimulated the secretion of cytokines and chemokines in both murine macrophages and human peripheral blood mononuclear cells. The most active compounds are 27 and 54. None of the compounds tested exhibited any significant cytostatic effect or antiviral effect.     

Modelling of Aniline-Vermiculite and Tetramethylammonium-Vermiculite; Test of Force Fields

Molecular mechanics simulations in Cerius² have been used for modelling vermiculite intercalated with tetramethylammonium and aniline cations. The published structure data obtained for these intercalated structures from X-ray single crystal diffraction have been used to test the force fields and modelling strategy for organo-clays. The strategy of modelling was based on the nonbond host-guest interactions and on rigid silicate layers and rigid guest species. The rigidity of silicate layers requires that the cell parameters a, b and % are kept fixed during the energy minimisation. The energy term was set up using the nonbond interaction terms only and the Crystal Packer module in Cerius² has been used for the energy minimisation. In Crystal Packer the rigid units, i.e. the silicate layers and guest species can be translated and rotated during energy minimisation and the cell parameters c, !, and # have been varied. Three sets of Van derWaals (VDW) parameters available in Crystal Packer: Tripos, Universal and Dreiding have been used in present molecular simulations. Ab initio MP2 calculations were performed to justify the application of the force field. The best agreement of molecular mechanics simulations with both: experimental and ab initio data was obtained with the Tripos VDW parameters for both intercalates. The results of modelling are in good agreement with the experimental data as to the cell parameters and the interlayer packing. The cell parameters reported by Vahedi-Faridi and Guggenheim (1997) for tetramethylammonium-vermiculite are: c = 13.616 Å, ! = 90°, # = 97.68° ; from the present modelling we obtained: c = 13.609 Å, ! = 90.19°, # = 97.56°. Tetramethylammonium-cations are arranged in one layer in the interlayer space. One C-C edge of NC₄ tetrahedra is perpendicular to the silicate layers. The deep immersion of the methyl groups into the ditrigonal cavities suggested by Vahedi-Faridi and Guggenheim was not confirmed by modelling. Slade and Stone (1984) presented the measured cell parameters for aniline vermiculite: c = 14.89 Å, ! = 90°, # = 97°; present result is: c = 14.81 Å, ! = 90.72°, # = 96.70° for partially exchanged vermiculite and c = 14.84 Å, ! = 90.53°, # = 97.17° for fully exchanged vermiculite. The aniline cations are positioned over the ditrigonal cavities alternating in their anchoring to lower and upper silicate layer. The C-N bonds are perpendicular to layers.     

Synthesis and immunobiological activity of base substituted 2-amino-3-(purin-9-yl)propanoic acid derivatives

2-Amino-3-(purin-9-yl)propanoic acids substituted at position 6 of the purine base moiety by dimethylamino, cyclopropylamino, pyrrolidin-1-yl, hydroxy, and sulfanyl group as well as their 2-aminopurine analogues were prepared from corresponding 9-(2,2-diethoxyethyl)purines and 2-aminopurines, respectively, by the Strecker synthesis. 2-Aminopropanoic acid derivatives were tested for their immunostimulatory and immunomodulatory potency. Some of these compounds significantly enhanced secretion of chemokines RANTES and MIP-1alpha, the most potent was 2-amino-6-sulfanylpurine derivative. Most of these compounds also augmented NO biosynthesis triggered primarily by IFN-gamma.     

Seasonal dynamics of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis as a response to abiotic factors and abundance of insect hosts

The seasonal dynamics of entomopathogenic nematodes (EPNs) of the genus Steinernema and Heterorhabditis were studied during one season in meadow and oak wood habitats, in the vicinity of Ceské Budejovice, Czech Republic. The influences of soil temperature, moisture, and abundance of suitable hosts on EPN dynamics were investigated. The host range of these nematodes, in both habitats was also observed. A total of four EPN species were found in both habitats. Steinernema affine was the dominant species both in oak wood and in meadow. Additionally, the oak wood habitat was inhabited by S. kraussei and S. weiseri; the meadow habitat by Heterorhabditis bacteriophora. The mean abundance of total EPN community was 28,000ind./m(2) in oak wood and 11,000ind./m(2) in meadow. The seasonal dynamics of entomopathogenic nematodes in both habitats were characterized by high nematode densities in the beginning of the season, followed by a rapid decrease, and then stabilization. EPN abundances did not show any apparent correlation with soil temperature and moisture, but they were negatively correlated with the abundance of suitable insect hosts. Inter- and intraspecific competition for limited nutrients (hosts) probably played a major role in EPN seasonal dynamics. Broad host range of entomopathogenic nematodes in both habitats was predominantly represented by dipteran and coleopteran larvae. Most common hosts belonged to the families Asilidae, Bibionidae, and Empididae (Diptera), as well as Carabidae and Curculionidae (Coleoptera).     

Containment of simian immunodeficiency virus infection in vaccinated macaques: correlation with the magnitude of virus-specific pre- and postchallenge CD4+ and CD8+ T cell responses

Macaques infected with the SIV strain SIVmac251 develop a disease closely resembling human AIDS characterized by high viremia, progressive loss of CD4(+) T cells, occurrence of opportunistic infection, cachexia, and lymphomas. We report in this study that vaccination with the genetically attenuated poxvirus vector expressing the structural Ags of SIVmac (NYVAC-SIV-gag, pol, env) in combination with priming with DNA-SIV-gag, env resulted in significant suppression of viremia within 2 mo after mucosal exposure to the highly pathogenic SIVmac251 in the majority of vaccinated macaques. The control of viremia in these macaques was long lasting and inversely correlated to the level of both pre- and postchallenge Gag-specific lymphoproliferative responses, as well as to the level of total SIV-specific CD4(+) T lymphocyte responses at the peak of acute viremia as detected by intracellular cytokine-staining assay. Viremia containment also correlated with the frequency of the immunodominant Gag(181-189)CM9 epitope-specific CD8(+) T cells present before the challenge or expanded during acute infection. These data indicate, for the first time, the importance of vaccine-induced CD4(+) Th cell responses as an immune correlate of viremia containment. The results presented in this work also further demonstrate the potential of a DNA-prime/attenuated poxvirus-boost vaccine regimen in an animal model that well mirrors human AIDS.     

Left ventricular diastolic filling in young persons with type 1 diabetes mellitus

The purpose of this study was to evaluate the influence of diabetes mellitus on left ventricular function. Using Doppler echocardiography we examined a group of 49 young (20-32 years old) persons with type 1 diabetes mellitus and a group of healthy age-matched men and evaluated the parameters of diastolic filling of the left ventricle (LV). We found significant differences in peak velocity of early filling of the left ventricle ((70.07+/-10.84 vers. 78.2+/-10.59 cm.s(-1), p+/-0. 01), peak velocity of late diastolic filling of LV (A = 47.74+/-11.6 vers. 43.15+/-7.48 cm.s(-1), p < or = 0.027), ratio E/A (1.53+/-0.4 vers. 1.84+/-0.33), time velocity integral of peak E (TVIE = 0.083+/-0.014 vers. 0.1+/-0.022 m, p < or = 0.001), time velocity integral of peak A (TVIA = 0.039+/-0.011 vers. 0.037+/-0.012 m, p < or = 0.3), ratio TVIE/TVIA (2.3+/-0.73 vers. 2.9+/-0.9, p < or = 0.001), time E (204.4+/-31.59 vers. 198.4+/-19.09 ms, p < or = 0.27), time A (126.9 < or = 23.0 vers. 113.5+/-15.59 ms, p < or = 0.002), time E/time A (1.64+/-0.3 vers. 1.76+/-0.22, p < or = 0.039) and duration of isovolumic relaxation period (IVRT = 88.2+/-10.8 vers. 71.13+/-8.4 ms, p < or = 0.0001). Despite significant differences all the results were in the range of values for the healthy population. However in detailed analysis we found that the values measured in young (20-32 years old) persons with type 1 diabetes mellitus corresponded with diastolic parameters of healthy men of the age of 50 years and more. Thus, diabetes mellitus can influence the relaxation properties of the left ventricle.     

Approaches to messenger RNA detection - comparison of methods

Detection of messenger RNA is an important part of current biomedical research, although utilized for decades. This communication endeavors to compare three most commonly used techniques of mRNA detection, i.e. Northern blot, ribonuclease protection assay (RPA), and real-time polymerase chain reaction (RT-PCR). Principles and general procedures of these methods are described, and advantages and weaknesses of each are discussed in terms of their specificity, sensitivity, difficulty, time and material demands as well as health and environmental risks. We conclude that choice of any method discussed depends on particular purpose, experience of the researcher, and on laboratory equipment and organization.     

Different methods of membrane domains isolation result in similar 2-D distribution patterns of membrane domain proteins.

Membrane domains are highly specialized parts of the cell plasma membrane, carrying on and augmenting the incoming signals. To study their structural and functional properties, it is crucial to find the least damaging mode of their isolation. Using two different cell lines, epithelial HEK cells (clone E2M11) and S49 lymphoma cells, three methods of membrane domain isolation (i.e., detergent extraction, alkaline treatment, and "drastic" homogenization) were tested for similarity and reproducibility by 2-D electrophoresis. Our data show that the protein composition of membrane domains obtained by different isolation methods is similar and that approximately 60% of the spots are present in all membrane domain preparations. Furthermore, the same degree of similarity of 2-D profiles of the most intensively silver stained spots found in membrane domains of the two cell lines derived from different tissues suggests that the composition of a large part of membrane domains proteins is conservative. We suggest that these proteins may either be involved in the organization of membrane domain structure or represent the conservative component of signal transduction machinery.     

Functional alteration of cytochrome c oxidase by SURF1 mutations in Leigh syndrome

Subacute necrotising encephalomyopathy (Leigh syndrome) due to cytochrome c oxidase (COX) deficiency is often caused by mutations in the SURF1 gene, encoding the Surf1 protein essential for COX assembly. We have investigated five patients with different SURF1 mutations resulting in the absence of Surf1 protein. All of them presented with severe and generalised COX defect. Immunoelectrophoretic analysis of cultured fibroblasts revealed 85% decrease of the normal-size COX complexes and significant accumulation of incomplete COX assemblies of 90-120 kDa. Spectrophotometric assay of COX activity showed a 70-90% decrease in lauryl maltoside (LM)-solubilised fibroblasts. In contrast, oxygen consumption analysis in whole cells revealed only a 13-31% decrease of COX activity, which was completely inhibited by detergent in patient cells but not in controls. In patient fibroblasts ADP-stimulated respiration was 50% decreased and cytofluorometry showed a significant decrease of mitochondrial membrane potential DeltaPsi(m) in state 4, as well as a 2.4-fold higher sensitivity of DeltaPsi(m) to uncoupler. We conclude that the absence of the Surf1 protein leads to the formation of incomplete COX complexes, which in situ maintain rather high electron-transport activity, while their H(+)-pumping is impaired. Enzyme inactivation by the detergent in patient cells indicates instability of incomplete COX assemblies.