The evaluation of vacA gene alleles frequency in Helicobacter pylori strains in children and adults in Podlaskie region

The frequency of Helicobacter pylori infection in population can depend on the organism resistance, genetic condition, and bacterial strains virulence. A vacA gene, of mosaic structure, which encodes vacuolating cytotoxin is one of the known genes of H. pylori. The existence of several different genotypes of s and m regions enables the formation of numerous combinations of vacA gene genome. The studies on vacA genotype revealed that the frequency of occurrence of H. pylori containing s1 or s2, as well as m1 and m2 alleles varies in different parts of the world. The aim of the studies performed in the group of children and adults was to evaluate the prevalence of particular vacA gene alleles distribution in the population of the Podlasie province. The allele s1, which occurred in 84.3% of the examined group (86.8% in children and 81.3% in adults), turned out to be the most frequently observed of the signal encoding region. Statistically significant differences in s1 and s2 alleles distribution in relation to a dwelling place were not detected. The allele m2 (42.1% in children and 59% in adults) was the allele of midregion, most frequently occurring in our studies. The allele m2 was observed more often in H. pylori strains in the inhabitants from the urban areas (data statistically significant).     

Intraspecific variability of cadmium tolerance in hydroponically grown Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.) seedlings

Indian mustard (Brassica juncea (L.) Czern.) is a promising plant species for phytoremediation of heavy metal polluted soils. However, genetic variability of metal tolerance in Indian mustard has not been studied. We evaluated intraspecific variation of Cd tolerance of this species by screening 64 varieties in hydroponics. The tolerance index (TI), calculated as percentage of root length of Cd-treated (7 μM CdCl₂) over untreated control seedlings, significantly varied from 34 to 79%, depending on the variety. Information about phenotypic and economic traits of the studied varieties was taken from the literature and subjected to a cluster analysis. The varieties were distributed into three clusters and most of the varieties characterized by the highest TI values (TI > 65%) were grouped together in one cluster. Moreover, TI negatively correlated with the following characteristics: yellow seed colour (R = −0.35, P = 0.005), total oil content (R = −0.33, P = 0.008), oleic acid (R = −0.25, P = 0.047) and linoleic acid (R = −0.36, P = 0.004) contents in seeds. The results showed the presence of significant variability for Cd tolerance in Indian mustard. The knowledge about correlations between Cd tolerance and phenotypic characteristics of plants might be utilized for rapid selection of cultivars to be used for phytoremediation of polluted soils.     

Morphology and Histochemistry of the Aesthetasc-Associated Epidermal Glands in Terrestrial Hermit Crabs of the Genus Coenobita (Decapoda: Paguroidea)

Crustaceans have successfully adapted to a variety of environments including fresh- and saltwater as well as land. Transition from an aquatic to a terrestrial lifestyle required adaptations of the sensory equipment of an animal, particularly in olfaction, where the stimulus itself changes from hydrophilic to mainly hydrophobic, air-borne molecules. Hermit crabs Coenobita spp. (Anomura, Coenobitidae) have adapted to a fully terrestrial lifestyle as adults and have been shown to rely on olfaction in order to detect distant food items. We observed that the specialized olfactory sensilla in Coenobita, named aesthetascs, are immersed in a layer of mucous-like substance. We hypothesized that the mucous is produced by antennal glands and affects functioning of the aesthetascs.Using various microscopic and histochemical techniques we proved that the mucous is produced by aesthetasc-associated epidermal glands, which we consider to be modified rosette-type aesthetasc tegumental glands known from aquatic decapods. These epidermal glands in Coenobita are multicellular exocrine organs of the recto-canal type with tubulo-acinar arrangement of the secretory cells. Two distinct populations of secretory cells were clearly distinguishable with light and electron microscopy. At least part of the secretory cells contains specific enzymes, CUB-serine proteases, which are likely to be secreted on the surface of the aesthetasc pad and take part in antimicrobial defense. Proteomic analysis of the glandular tissue corroborates the idea that the secretions of the aesthetasc-associated epidermal glands are involved in immune responses.We propose that the mucous covering the aesthetascs in Coenobita takes part in antimicrobial defense and at the same time provides the moisture essential for odor perception in terrestrial hermit crabs. We conclude that the morphological modifications of the aesthetasc-associated epidermal glands as well as the functional characteristics of their secretions are important adaptations to a terrestrial lifestyle.     

Mass spectral characterization of organophosphate-labeled,tyrosine-containing peptides: Characteristic mass fragments and a new binding motif for organophosphates

We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8–8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.     

Agmatine stimulates hepatic fatty acid oxidation: a possible mechanism for up-regulation of ureagenesis

We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled beta-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled beta-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial beta-oxidation and suggest a coupling between the stimulation of hepatic beta-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.     

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the gamma-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Abeta). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.     

Assaying the probabilities of obtaining maternally inherited heteroplasmy as the basis for modeling OXPHOS diseases in animals

Gross alterations in cell energy metabolism underlie manifestations of hereditary OXPHOS (oxidative phosphorylation) diseases, many of which depend on proportion of mutant mitochondrial DNA (mtDNA) in tissues. An animal model of OXPHOS disease with maternal inheritance of mitochondrial heteroplasmy might help understanding the peculiarities of abnormal mtDNA distribution and its effect on pre- and postnatal development. Previously we obtained mice that carry human mtDNA in some tissues. It co-existed with murine mtDNA (heteroplasmy) and was transmitted maternally to the progeny of animals developed from zygotes injected with human mitochondria. To analyze the probability of obtaining heteroplasmic mice we increased the number of experiments with early embryos and obtained more specimens from F1. About 33% of zygotes injected with human mtDNA developed into post-implantation embryos (7th-13th days). Lower amount of such developed into neonate mice (ca. 21%). Among post-implantation embryos and in generations F0 and F1 percentages of human mtDNA-carriers were ca. 14-16%. Such percentages are sufficient for modeling maternally inherited heteroplasmy in small animal groups. More data are needed to understand the regularities of anomalous mtDNA distribution among cells and tissues and whether heart and muscles frequently carrying human mtDNA in our experiments are particularly susceptible to heteroplasmy.     

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.     

Beneficial metabolic effects of M3 muscarinic acetylcholine receptor deficiency

Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.