2′, 3′-<Emphasis Type="Italic">O</Emphasis>-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y<Subscript>2</Subscript> nucleotide receptor

In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X₇ receptor activity. We show here that in the absence of extracellular Ca²⁺, BzATP-generated increase in [Ca²⁺]_i via Ca²⁺ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca²⁺ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X₇ receptor, did not reduce any of the two components of the Ca²⁺ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca²⁺ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y₂ but not the P2X₇ receptors.     

Occurrence of 16s rRNA methylase ArmA producing Enterobacteriaceae in a general hospital in Warsaw, Poland

Resistance to gentamicin, amikacin and kanamycin was screened in 270 clinical isolates of Enterobacteriaceae originated from April 19 to May 19, 2010 in a regular hospital in Warsaw, Poland. Most of the isolated bacteria were considered pathogenic. Nineteen isolates (7%) were simultaneously resistant to two or three of the tested aminoglycosides. MICs of the three aminoglycosides ranged form 128 to 1024 mcg/ml for six isolates. These isolates were suspected to produce 16S rRNA methylase. Genes encoding for three methylases reported in Europe: ArmA, RmtB and RmtC were searched by PCR. The armA gene was detected in all of the six isolates. This group encompassed Enterobacter cloacae (n=4), Klebsiella pneumoniae (n=1) and Proteus mirabilis (n=1). Five isolates of this group carried the bla(CAX-M) gene for CTX-M type ESBL. The remaining isolate E. cloacae DM0340 was ESBL negative and lacked bla(CRX-M) that may suggest an altered genetic environment of the armA gene in this isolate. Our results showed that 2.2% of the tested isolates produced 16S rRNA methylase ArmA. This finding may argue for a high incidence of ArmA producing Enterobacteriaceae in Poland when compared to reports from other European countries.     

Effects of MMP-9 inhibition by doxycycline on proteome of lungs in high tidal volume mechanical ventilation-induced acute lung injury

Background  

Although mechanical ventilation (MV) is a major supportive therapy for patients with acute respiratory distress syndrome, it may result in side effects including lung injury. In this study we hypothesize that MMP-9 inhibition by doxycycline might reduce MV-related lung damage. Using a proteomic approach we identified the pulmonary proteins altered in high volume ventilation-induced lung injury (VILI). Forty Wistar rats were randomized to an orally pretreated with doxycycline group (n = 20) or to a placebo group (n = 20) each of which was followed by instrumentation prior to either low or high tidal volume mechanical ventilation. Afterwards, animals were euthanized and lungs were harvested for subsequent analyses.     

Effect of Garlic Supplementation on Erythrocytes Antioxidant Parameters, Lipid Peroxidation, and Atherosclerotic Plaque Formation Process in Oxidized Oil-Fed Rabbits

Effect of garlic supplementation on blood antioxidant status, lipid peroxidation, and coronary plaque formation process was investigated in oxidized oil-fed rabbits. Eighteen adult male mixed European rabbits were given a balanced diet (21 g% protein, 34 g% fat, 45 g% carbohydrate), which contained isocaloristic addition of nonoxidized or oxidized rapeseed oil in the presence and absence of garlic. The experiment lasted 24 weeks. At the beginning and every 6 weeks, rabbits were weighed, and blood was taken. To evaluate the antioxidant status of the rabbits, erythrocytes malondialdehyde (MDA) concentration, total superoxide dismutase (t-SOD), and glutathione peroxidase (GPX) activations were determined. After the experiment was completed, aortas were dissected for histological examinations. Changes in the contents of the above parameters and histological examinations showed that oxidized rapeseed, oil administered to rabbits, caused the development of atherosclerotic changes and disturbed antioxidant status. The addition of garlic in such diets inhibited atherosclerotic changes in the aorta wall, and it is related to the homeostatic activity of antioxidative enzymes and lipid peroxidation.     

The effect of luminal ghrelin on pancreatic enzyme secretion in the rat

Ghrelin, a 28-amino-acid peptide produced predominantly by oxyntic mucosa has been reported to affect the pancreatic exocrine function but the mechanism of its secretory action is not clear. The effects of intraduodenal (i.d.) infusion of ghrelin on pancreatic amylase outputs under basal conditions and following the stimulation of pancreatic secretion with diversion of pancreato-biliary juice (DPBJ) as well as the role of vagal nerve, sensory fibers and CCK in this process were determined. Ghrelin given into the duodenum of healthy rats at doses of 1.0 or 10.0 microg/kg increased pancreatic amylase outputs under basal conditions or following the stimulation of pancreatic secretion with DPBJ. Bilateral vagotomy as well as capsaicin deactivation of sensory fibers completely abolished all stimulatory effects of luminal ghrelin on pancreatic exocrine function. Pretreatment with lorglumide, a CCK(1) receptor blocker, reversed the stimulation of amylase release produced by intraduodenal application of ghrelin. Intraduodenal ghrelin at doses of 1.0 or 10.0 microg/kg increased plasma concentrations of CCK and ghrelin. In conclusion, ghrelin given into the duodenum stimulates pancreatic enzyme secretion. Activation of vagal reflexes and CCK release as well as central mechanisms could be implicated in the stimulatory effect of luminal ghrelin on the pancreatic exocrine functions.     

Peptidomic analysis of skin secretions from Rana heckscheri and Rana okaloosae provides insight into phylogenetic relationships among frogs of the Aquarana species group

The members of the Aquarana (or Rana catesbeiana species group) form a monophyletic group comprising seven species: R. catesbeiana, Rana clamitans, Rana grylio, Rana virgatipes, Rana septentrionalis, Rana heckscheri and Rana okaloosae. Previous work has led to structural characterization of the antimicrobial peptides present in electrically-stimulated skin secretions from the first five species listed and this study presents the primary structures of orthologs from the river frog R. heckscheri and the Florida bog frog R. okaloosae. Peptidomic analysis of R. heckscheri and R. okaloosae skin secretions led to the identification of peptides with antimicrobial activity belonging to the ranalexin, ranatuerin-2, and temporin families. In addition, a peptide (GFLDIIKDTGKDFAVKILNNLKCKLAGGCPR) was isolated from R. okaloosae whose primary structure identified it as a member of the palustrin-2 family. Consistent with previous data based upon morphological analysis and comparisons of the nucleotide sequences of mitochondrial and ribosomal genes, cladistic analysis based upon a comparison of the amino acid sequences of antimicrobial peptides indicates a sister-group relationship between R. heckscheri and R. grylio and a close, but less well defined, phylogenetic relationship between R. okaloosae and R. clamitans.     

New face of antiapoptotic proteins. I. Protein Mcl-1

Main regulators of apoptosis belong to Bcl-2 protein family and apoptosis inhibitory proteins--IAPs. In this review the apoptosis inhibitor--Mcl-1 protein is profoundly characterized. It is important that this unique short-living protein--the member of Bcl-2 family may also operate as apoptosis promoting agent, which results of alternative splicing of its pre-mRNA, posttranslational modifications or proteolysis. The review presents also other functions of Mcl-1, i.e. involvement in cell cycle regulation, elongation of telomers. Elevated expression of Mcl-1 accompanies the development of various cancers, neurodegenerative disorders and also infectious diseases. The obtained results indicate that expression level of Mcl-1 may be useful in treatment decisions of large number of diseases. Ablating expression of this protein may be an attractive therapeutic strategy in the treatment of various cancers, and the diseases where Mcl-1 may play a key role in apoptosis supression.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

Effect of exercise training on cardiac oxytocin and natriuretic peptide systems in ovariectomized rats

Exercise training results in cardiovascular and metabolic adaptations that may be beneficial in menopausal women by reducing blood pressure, insulin resistance, and cholesterol level. The adaptation of the cardiac hormonal systems oxytocin (OT), natriuretic peptides (NPs), and nitric oxide synthase (NOS) in response to exercise training was investigated in intact and ovariectomized (OVX) rats. Ovariectomy significantly augmented body weight (BW), left ventricle (LV) mass, and intra-abdominal fat pad weight and decreased the expression of oxytocin receptor (OTR), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and guanylyl cyclase-A (GC-A), in the right atrium (RA) and LV, indicating estrogenic control of these genes. These effects of ovariectomy were counteracted by 8-wk-long exercise training which decreased fat pad weight (33.4 +/- 2.3 to 23.4 +/- 3.1 g, n = 8, P < 0.05), plasma free fatty acids (0.124 +/- 0.033 to 0.057 +/- 0.010 mM, n = 8, P < 0.01), and plasma triacylglycerol (0.978 +/- 0.174 to 0.588 +/- 0.115 mM, n = 8, P < 0.05). Chronic exercise tended to decrease BW and stimulated ANP (4- to 5-fold) and OTR gene expression in the LV and RA and BNP and inducible NOS (iNOS) mRNA in the LV. In sham-operated rats, exercise augmented ANP expression in the RA, downregulated GC-A mRNA in the LV and RA, but increased its expression threefold in the RA of OVX animals. Endothelial NOS and iNOS expression was enhanced in the left atrium of sham-operated rats. Altogether, these data indicate that in OVX animals, chronic exercise significantly enhances cardiac OT, NPs, and NOS, thus implicating all three hormonal systems in the beneficial effects of exercise training.