Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane

Albeit previous experiments suggest potential anti-inflammatory effect of exogenous methane (CH₄) in various organs, the mechanism of its bioactivity is not entirely understood. We aimed to investigate the potential mitochondrial effects and the underlying mechanisms of CH₄ in rat cardiomyocytes and mitochondria under simulated ischaemia/reperfusion (sI/R) conditions. Three-day-old cultured cardiomyocytes were treated with 2.2% CH₄-artificial air mixture during 2-hour-long reoxygenation following 4-hour-long anoxia (sI/R and sI/R + CH₄, n = 6-6), with normoxic groups serving as controls (SH and SH + CH₄; n = 6-6). Mitochondrial functions were investigated with high-resolution respirometry, and mitochondrial membrane injury was detected by cytochrome c release and apoptotic characteristics by using TUNEL staining. CH₄ admixture had no effect on complex II (CII)-linked respiration under normoxia but significantly decreased the complex I (CI)-linked oxygen consumption. Nevertheless, addition of CH₄ in the sI/R + CH4 group significantly reduced the respiratory activity of CII in contrast to CI and the CH₄ treatment diminished mitochondrial H₂O₂ production. Substrate-induced changes to membrane potential were partially preserved by CH₄, and additionally, cytochrome c release and apoptosis of cardiomyocytes were reduced in the CH₄-treated group. In conclusion, the addition of CH₄ decreases mitochondrial ROS generation via blockade of electron transport at CI and reduces anoxia-reoxygenation-induced mitochondrial dysfunction and cardiomyocyte injury in vitro.     

Dishevelled links basal body docking and orientation in ciliated epithelial cells

Some epithelia contain cells with multiple motile cilia that beat in a concerted manner. New tools and experimental systems have facilitated molecular studies of cilium biogenesis and the coordinated planar polarization of cilia that leads to their concerted motility. A recent elegant study using embryonic frog epidermis demonstrates that Dishevelled, a key regulator of both the Wnt-beta-catenin and planar cell polarity pathways, controls both the docking and planar polarization of ciliary basal bodies.     

gamma-Tocopherol nebulization by a lipid aerosolization device improves pulmonary function in sheep with burn and smoke inhalation injury

Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.     

Prevalent role of Akt and ERK activation in cardioprotective effect of Ca2+ channel- and beta-adrenergic receptor blockers

We studied cardioprotective as well as Akt and extracellular signal-activated kinase (ERK) activating effect of a Ca(2+) antagonist and a beta-adrenergic receptor blocker during ischemia-reperfusion, and compared these properties of the substances with that of a poly(ADP-ribose) polymerase (PARP) inhibitor used as a positive control throughout the experiments. Langendorff-perfused isolated rat hearts were subjected to 25 min global ischemia followed by 45 min reperfusion, and recovery of energy metabolism as well as functional cardiac parameters were monitored. Although to varying extents, all substances improved recovery of creatine phosphate, ATP, intracellular pH, and reutilization of inorganic phosphate. These favorable changes were accompanied by improved recovery of heart function parameters and reduced infarct size. In addition and again to varying extents, all studied substances decreased oxidative damage (lipid peroxidation and protein oxidation), and activated Akt, glycogen synthase kinase (GSK)-3beta, and ERK1/2. Correlation between cardioprotective and kinase activating effectivity of the compounds proved to be statistically significant. Physiological significance of these kinase activations was established by demonstrating that inhibition of Akt by LY294002 and ERK1/2 by PD98059 compromised the cardioprotective effect of all the substances studied. In conclusion, we demonstrated for the first time that activation of phosphatidylinositol-3-kinase (PI-3K)-Akt and ERK2 pathways significantly contributed to cardioprotective effects of a Ca(2+) antagonist and a beta-adrenergic receptor blocker. Furthermore, we found a strong correlation between cardioprotective and kinase-activating potencies of the substances studied (Verapamil, Metoprolol and two PARP inhibitors), which indicated the potentiality of these kinases as drug-targets in the therapy of ischemic heart disease.     

Effect of different concentrations of serotonin, histamine and insulin on the hormone (serotonin and ACTH) production of Tetrahymena in nutrient-free physiological milieu

Cell populations of Tetrahymena pyriformisGL were kept in nutrient-free (Losina) milieu and treated with different (10⁻⁶–10⁻²¹ M) concentrations of serotonin, histamine or insulin for 30 min. Following that the hormone (serotonin and adrenocorticotropin (ACTH) content of the cells were measured by immunocytochemical flow cytometric method. Serotonin reduced histamine when applied in 10⁻¹² and 10⁻¹⁵ M concentrations, while elevated ACTH levels when applied in 10⁻⁶, 10⁻⁹ and 10⁻²¹ M concentrations. Histamine reduced serotonin concentration at 10⁻⁹–10⁻²¹ M concentrations and increased ACTH in 10⁻⁶ M. Insulin elevated both hormones’ content in each concentration except at 10⁻¹² M. The results demonstrate that (1) in nutrient-free conditions the hormonal effects differ from that of nutrient-rich (tryptone + yeast) condition; (2) there is an optimal hormone concentration, which causes the strongest effect and this is different for each hormones; (3) the hormone receptors of Tetrahymena are very sensitive; as they react to zeptomolar concentrations. Such small concentration is even more effective than higher ones. Since hormones must become highly diluted in the natural environment of Tetrahymena, it seems that such low concentrations are the actual physiological concentrations.     

A novel human polyomavirus closely related to the african green monkey-derived lymphotropic polyomavirus

We identified a novel human polyomavirus from a kidney transplant patient under immunosuppressive treatment, by use of a generic PCR. The genome of the virus was completely amplified and sequenced. In phylogenetic analyses, it appeared as the closest relative to the African green monkey-derived lymphotropic polyomavirus (LPV). Further investigation of clinical samples from immunocompromised patients with specific nested PCR revealed additional positive samples, indicating that the virus naturally infects humans. The virus was tentatively named human polyomavirus 9 (HPyV9). The previously observed seroreactivity to LPV in human populations might find a partial explanation in the circulation of HPyV9.     

Genetic Heterogeneity Reveals On-Going Speciation and Cryptic Taxonomic Diversity of Stream-Dwelling Gudgeons (Teleostei,Cyprinidae) in the Middle Danubian Hydrosystem (Hungary)

Although stream-dwelling gudgeons (Cyprinidae, genus: Gobio) are widespread in Central Europe, the taxonomy of this group and the distribution of its species are still unexplored in detail. The aims of our study are to ascertain taxonomic composition and distribution of the former Gobio gobio superspecies in the inner area of the Carpathian Basin. Since the presence of cryptic species is suspected in this area, we examined the taxonomic and phylogenetic relationships of Central European Gobio taxa by sequencing the mitochondrial DNA control region (mtCR). Additionally, we characterized the genetic structure of 27 stream-dwelling gudgeon populations of this area by Amplified Fragment Length Polymorphism (AFLP). Results of mtCR analysis proved the presence of three species already known as G. obtusirostris (dominant in NW-Hungary), G. gobio (sporadic) and G. carpathicus (sporadic). Additionally, the analysis revealed the existence of one doubtful taxon, G. sp1 (dominant in NE-Hungary), and a new isolated haplogroup (dominant in SW-Hungary). Although Network analysis showed significant detachment among haplogroups, their genetic distances were quite small. Therefore Bayesian phylogenetic analysis showed weak nodal support for the branching pattern both for newly described haplotypes, and for the already accepted species. AFLP data showed distinct population structure and a clear pattern of isolation was revealed by distance of stocks. At the same time, level of separation was not affected by the altitudinal position of sites. Moreover we found three major clusters of populations which were separated according to hydrographic regions, and corresponded to the findings of mtCR analysis. Our results suggest the on-going speciation of gudgeons in the Carpathian Basin, however the separation of haplogroups seems to only be an intermediate phase. The discovered natural pattern seems to be only slightly influenced by anthropogenic impacts. Additionally our results put into question the suitability of the recently accepted within Gobio genus taxonomy.     

Unusual DNA structures at the integration site of an HIV provirus

Supercoiled pHXBc2 DNA (containing the genome of the human immunodeficiency virus type 1 and human sequences) migrated more slowly than linear DNA in native and ethidium bromide agarose gel electrophoresis at 4.5 volts/cm, suggesting the presence of unusual DNA structures. S1 nuclease analysis of pHXBc2 revealed two S1 hypersensitive sites. Site I was located within a 25 bp direct repeat in host DNA 0.6 kB upstream from the 5' LTR. Site II was mapped 0.2 kB upstream from the vif gene start site. Sequence analysis showed that Site I sequences could assume different unusual DNA structures, whereas sequences at Site II could assume either slipped or H-DNA forms. Unusual DNA structures in host DNA may be associated with active chromatin regions and may favor proviral integration.     

Synergistic Neutralization of Simian-Human Immunodeficiency Virus SHIV-vpu+ by Triple and Quadruple Combinations of Human Monoclonal Antibodies and High-Titer Anti-Human Immunodeficiency Virus Type 1 Immunoglobulins

We have tested triple and quadruple combinations of human monoclonal antibodies (MAbs), which are directed against various epitopes on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, and a high-titer anti-HIV-1 human immunoglobulin (HIVIG) preparation for their abilities to neutralize a chimeric simian-human immunodeficiency virus (SHIV-vpu⁺). This virus encodes the HIV-1 strain IIIB env, tat, rev, and vpu genes. The quantitative nature of the Chou-Talalay method (Adv. Enzyme Regul. 22:27–55, 1984) allows ranking of various combinations under identical experimental conditions. Of all triple combinations tested, the most potent neutralization was seen with MAbs 694/98D plus 2F5 plus 2G12 (directed against domains on V3, gp41, and gp120, respectively) as measured by the total MAb concentration required to reach 90% neutralization (90% effective concentration [EC₉₀], 2.0 μg/ml). All triple combinations involving MAbs and/or HIVIG that were tested yielded synergy with combination index values of <1; the dose reduction indices (DRIs) ranged from 3.1 to 26.2 at 90% neutralization. When four MAbs (the previous three plus MAb F105, directed against the CD4 binding site) were combined, higher neutralization potency (EC₉₀, 1.8 μg/ml) and a higher degree of synergy compared to any triple combination were seen. The mean DRIs of the quadruple combination were approximately twice that of the most synergistic triple combination. We conclude that human MAbs targeting different HIV-1 envelope glycoprotein epitopes exhibit strong synergy when used in combination, a fact that could be exploited clinically for passive immunoprophylaxis against HIV-1.