Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.     

Kinetics of benzo(a)pyrene hydroxylation at varying concentrations of NADPH and NADH in liver microsomes of nontreated and beta-naphthoflavone-treated C57BL/2 and DBA/2 mice

The biphasic kinetics of hydroxylation of benzo(a)pyrene (B(a)P) dependent on the concentration of either NADPH or NADH has been observed in DBA/2 (AhdAhd) but not in C57BL/6 (AhbAhb) beta-naphthoflavone-treated mice. On the other hand, in nontreated mice, this biphasic kinetics has been observed in both strains of mice. This shows that characteristics of biphasic kinetics differentiate Ahb from Ahd mice only after treatment with methylcholanthrene-type of inducers. The discussed biphasic kinetics was more regular and distinct in the NADH-dependent reaction as compared with NADPH-dependent hydroxylation of B(a)P. It is suggested that the NADH-specific pathway of electron transport to cytochrome P-450 is necessary for the occurrence of this effect both in NADH- and NADPH supported reaction.     

Mutations in KPTN Cause Macrocephaly,Neurodevelopmental Delay,and Seizures

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.     

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.     

Oxidized low-density lipoprotein (oxLDL) plasma levels and oxLDL to LDL ratio — Are they real oxidative stress markers in dialyzed patients?

AimsDyslipidemia and oxidative stress are commonly present in patients during maintenance dialysis treatment. However, the significance of oxidized LDL (oxLDL) as a marker of oxidative stress in uremia is still unresolved. The aim of this study was to establish the role of oxLDL and oxLDL/LDL ratio as markers of lipoprotein abnormalities and oxidative stress in the dialyzed patients.Main methodsPlasma oxLDL level was measured by ELISA, and oxLDL/LDL ratio was calculated in 106 dialyzed patients and 20 controls. The linkages between oxLDL, oxLDL/LDL ratio and lipid profile and oxidative stress markers malondialdehyde (MDA) and Cu/Zn superoxide dismutase (Cu/Zn SOD) levels were also analyzed.Key findingsOxLDL levels and oxLDL/LDL ratio were similar in hemodialyzed patients and controls, whereas these parameters were lower in peritoneally dialyzed patients when compared to healthy individuals. In contrast, both MDA and Cu/Zn SOD levels were significantly higher in uremics than in controls. oxLDL and oxLDL/LDL ratio positively correlated with lipid profile (except of HDL), whereas there were no positive associations between these parameters and both MDA and Cu/Zn SOD. Multiple regression analysis confirmed that increased oxLDL/HDL and TC/HDL ratios and total cholesterol levels are the parameters which independently predicted oxLDL in dialyzed patients. In the case of oxLDL/LDL ratio, the independent variables were oxLDL/HDL ratio, total cholesterol and HDL levels.SignificanceoxLDL levels and oxLDL/LDL ratio seem to be the markers of lipoprotein abnormalities rather than the markers of oxidative stress in the population of dialyzed patients.