19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.     

Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on Escherichia coli survival

In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO.     

Identification and expression of two novel cytochrome P450 genes, belonging to CYP4 and a new CYP331 family, in the polychaete Capitella capitata sp.I

The polychaete Capitella capitata sp.I has a high capacity to metabolize polycyclic aromatic hydrocarbons (PAHs) which are among the most hazardous environmental pollutants with significant biological effects. In the present study, two novel cytochrome P450 (CYP) genes were identified in this species. One was named CYP331A1, the first member of a new family of CYP331, and the other CYP4AT1 is the first member of a new subfamily CYP4AT. Both of these genes are constitutively expressed in the worms and detectable by RT-PCR. The expression of CYP331A1 mRNA was observed to be more sensitive to PAH exposure than CYP4AT1, which indicated that CYP331A1 should play a more important role than CYP4AT1 in PAH metabolism in this species. Considering the importance of C. capitata sp.I in taking up PAH and other organic pollutants from contaminated marine sediments with the potential for subsequent food-chain transfer, our results are important for understanding the molecular basis of biotransformation and detoxification in this invertebrate, and also have evolutionary significance for understanding the diversity and history of the CYP superfamily.     

Overlapping Messages and Survivability

The phenomenon of overlapping of various sequence messages in genomes is a puzzle for evolutionary theoreticians, geneticists, and sequence researchers. The overlapping is possible due to degeneracy of the messages, in particular, degeneracy of codons. It is often observed in organisms with a limited size of genome, possessing polymerases of low fidelity. The most accepted view considers the overlapping as a mechanism to increase the amount of information per unit length. Here we present a model that suggests direct evolutionary advantage of the message overlapping. Two opposing drives are considered: (a) reduction in the amount of vulnerable points when the overlapping of two messages involves common critical points and (b) cumulative compromising cost of coexistence of messages at the same site. Over a broad range of conditions the reduction of the target size prevails, thus making the overlapping of messages advantageous.[Reviewing Editor: Dr. Martin Kreitman]     

Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of inflammation

Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4. In this study, we show that half-maximal inhibition of LPS-induced elevation of E-selectin mRNA in endothelial cells developed at concentrations of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) 10-fold lower than those required to induce proinflammatory response. Similar concentration difference was observed for other classes and molecular species of OxPLs. Upon injection into mice, OxPAPC did not elevate plasma levels of IL-6 and keratinocyte chemoattractant but strongly inhibited LPS-induced upregulation of these inflammatory cytokines. Thus, both in vitro and in vivo, anti-LPS effects of OxPLs are observed at lower concentrations than those required for their proinflammatory action. Quantification of the most abundant oxidized phosphatidylcholines by HPLC/tandem mass spectrometry showed that circulating concentrations of total oxidized phosphatidylcholine species are close to the range where they demonstrate anti-LPS activity but significantly lower than that required for induction of inflammation. We hypothesize that low levels of OxPLs in circulation serve mostly anti-LPS function and protect from excessive systemic response to TLR4 ligands, whereas proinflammatory effects of OxPLs are more likely to develop locally at sites of tissue deposition of OxPLs (e.g., in atherosclerotic vessels).     

Infection by Trypanosoma cruzi Enhances Anion Conductance in Rat Neonatal Ventricular Cardiomyocytes

Recent studies on malaria-infected erythrocytes have shown increased anion channel activity in the host cell membrane, increasing the exchange of solutes between the cytoplasm and exterior. In the present work, we addressed the question of whether another intracellular protozoan parasite, Trypanosoma cruzi, alters membrane transport systems in the host cardiac cell. Neonatal rat cardiomyocytes were cultured and infected with T. cruzi in vitro. Ion currents were measured by patch-clamp technique in the whole-cell configuration. Two small-magnitude instantaneous anion currents, outward- and inward-rectifying, were recorded in all noninfected cardiomyocytes. In addition, ~10% of cardiomyocytes expressed a large anion-preferable, time-dependent current activated at positive membrane potentials. Hypotonic (230?mOsm) treatment resulted in the disappearance of the time-dependent current but provoked a dramatic increase of the instantaneous outward-rectifying one. Both instantaneous currents were suppressed by intracellular Mg(2+). T. cruzi infection did not provoke new anion currents in the host cells but caused an increase of the density of intrinsic swelling-activated outward current, up to twice in heavily infected cells. The occurrence of a time-dependent current dramatically increased in infected cells in the presence of Mg(2+) in the intracellular solution, from ~10 to ~80%, without a significant change of the current density. Our findings represent one further, besides the known Plasmodium falciparum, example of an intracellular parasite which upregulates the anionic currents expressed in the host cell.     

Reciprocal chromosome painting between three laboratory rodent species

The laboratory mouse (Mus musculus, 2n = 40), the Chinese hamster (Cricetulus griseus, 2n = 22), and the golden (Syrian) hamster (Mesocricetus auratus, 2n = 44) are common laboratory animals, extensively used in biomedical research. In contrast with the mouse genome, which was sequenced and well characterized, the hamster species has been set aside. We constructed a chromosome paint set for the golden hamster, which for the first time allowed us to perform multidirectional chromosome painting between the golden hamster and the mouse and between the two species of hamster. From these data we constructed a detailed comparative chromosome map of the laboratory mouse and the two hamster species. The golden hamster painting probes revealed 25 autosomal segments in the Chinese hamster and 43 in the mouse. Using the Chinese hamster probes, 23 conserved segments were found in the golden hamster karyotype. The mouse probes revealed 42 conserved autosomal segments in the golden hamster karyotype. The two largest chromosomes of the Chinese hamster (1 and 2) are homologous to seven and five chromosomes of the golden hamster, respectively. The golden hamster karyotype can be transformed into the Chinese hamster karyotype by 15 fusions and 3 fissions. Previous reconstructions of the ancestral murid karyotype proposed diploid numbers from 2n = 52 to 2n = 54. By integrating the new multidirectional chromosome painting data presented here with previous comparative genomics data, we can propose that syntenies to mouse Chrs 6 and 16 were both present and to hypothesize a diploid number of 2n = 48 for the ancestral Murinae/Cricetinae karyotype.     

Primordia Vita. Deconvolution from Modern Sequences.

Evolution of the triplet code is reconstructed on the basis of consensus temporal order of appearance of amino acids. Several important predictions are confirmed by computational sequence analyses. The earliest amino acids, alanine and glycine, have been encoded by GCC and GGC codons, as today. They were succeeded, respectively, by A- and G-series of amino acids, encoded by pyrimidine-central and purine-central codons. The length of the earliest proteins is estimated to be 6–7 residues. The earliest mRNAs were short G+C-rich molecules. These short sequences could have formed hairpins. This is confirmed by analysis of modern prokaryotic mRNA sequences. Predominant size of detected ancient hairpins also corresponds to 6–7 amino acids, as above. Vestiges of last common ancestor can be found in extant proteins in form of entirely conserved short sequences of size six to nine residues present in all or almost all sequenced prokaryotic proteomes (omnipresent motifs). The functions of the topmost conserved octamers are not involved in the basic elementary syntheses. This suggests an initial abiotic supply of amino acids, bases and sugars. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Childhood thyroid cancer, radiation dose from Chernobyl, and dose uncertainties in Bryansk Oblast, Russia: a population-based case-control study

A population-based case-control study was conducted to estimate the radiation-related risk of thyroid cancer in persons who were exposed in childhood to (131)I from the Chernobyl accident of April 26, 1986 and to investigate the impact of uncertainties in individual dose estimates. Included were all 66 confirmed cases of primary thyroid cancer diagnosed from April 26, 1986 through September 1998 in residents of Bryansk Oblast, Russia, who were 0-19 years old at the time of the accident, along with two individually matched controls for each case. Thyroid radiation doses, estimated using a semi-empirical model based on environmental contamination data and individual characteristics, ranged from 0.00014 Gy to 2.73 Gy and had large uncertainties (median geometric standard deviation 2.2). The estimated excess relative risk (ERR) associated with radiation exposure, 48.7/Gy, was significantly greater than 0 (P = 0.00013) but had an extremely wide 95% confidence interval (4.8 to 1151/Gy). Adjusting for dose uncertainty nearly tripled the ERR to 138/Gy, although this was likely an overestimate due to limitations in the modeling of dose uncertainties. The radiation-related excess risk observed in this study is quite large, especially if the uncertainty of dose estimation is taken into account, but is not inconsistent with estimates previously reported for risk after (131)I exposure or acute irradiation from external sources.