Metabolic footprint of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> NCFM at different pH

Lactobacillus acidophilus NCFM is a well known microorganism from the genomic and probiotic point of view. In order to analyze the potential interactions of NCFM with the surrounding environment, in vitro tests with the metabolic footprinting approach were performed. It was found that NCFM increased the concentration of lactic acid, succinic acid, adenine and arginine in the medium. The metabolism of NCFM did not change significantly between pH 5 and 7, suggesting that other environmental factors than pH might have bigger impact on its colonization throughout the gastrointestinal tract.     

Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.     

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10^?9 nor 10^?6 mol/L concentration. Ghrelin (10^?6 mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10^?6 mol/L ghrelin increased, while 10^?4 mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.     

Coordination of iron ions in the form of histidinyl dinitrosyl complexes does not prevent their genotoxicity

Formation of dinitrosyl iron complexes (DNICs) was observed in a wide spectrum of pathophysiological conditions associated with overproduction of NO. To gain insight into the possible genotoxic effects of DNIC, we examined the interaction of histidinyl dinitrosyl iron complexes (HIS-DNIC) with DNA by means of circular dichroism. Formation of DNIC was monitored by EPR and FT/IR spectroscopy. Vibrational bands for aquated HIS-DNIC are reported. Dichroism results indicate that HIS-DNIC changes the conformation of the DNA in a dose-dependent manner in 10 mM phosphate buffer (pH 6). Increase of the buffer pH or ionic strength decreased the effect. Comparison of HIS-DNIC DNA interaction with the effect of hydrated Fe²⁺ ion revealed many similarities. The importance of iron ions in HIS-DNIC induced genotoxicity is confirmed by plasmid nicking assay. Treatment of pUC19 plasmid with 1 μM HIS-DNIC did not affect the plasmid supercoiling. Higher concentrations of HIS-DNIC induced single strand breaks. The effect was completely abrogated by addition of deferoxamine, a specific strong iron chelator. Our data reveal that formation of HIS-DNIC does not prevent DNA from iron-induced damage and imply that there is no direct interrelationship between iron–NO coordination and their mutual toxicity modulation.     

The mechanism of autocatalytic activation of plant-type L-asparaginases

Plant l-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (alpha and beta) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit beta. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr(179) side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.     

Growth hormone therapy in boy with panhypopituitarism may induce pilomatricoma recurrence: case report

Pilomatricoma is usually a solitary subcutaneous nodule. Recurrence of the nodule after surgical excision is very rare. Pilomatricoma occurrence in patients with growth hormone (GH) deficiency has not been reported, yet. We report a 14-year-old boy with pilomatricoma and panhypopituitarism. After GH therapy had been started, we observed two relapses of previously completely excised pilomatricoma in the same location and a new pilomatricoma formation on the chin.     

Variability in benthic diazotrophy and cyanobacterial diversity in a tropical intertidal lagoon

Benthic nitrogen fixation has been estimated to contribute 15 Tg N year(-1) to the marine nitrogen budget. With benthic marine nitrogen fixation being largely overlooked in more recent surveys, a refocus on benthic diazotrophy was considered important. Variations in nitrogenase activity (acetylene reduction-gas chromatography) in a tropical lagoon in the western Indian Ocean (Zanzibar, Tanzania) were monitored over a 3-year period (2003-2005) and related to cyanobacterial and diazotrophic microbial diversity using a polyphasic approach. Different nitrogenase activity patterns were discerned, with the predominant pattern being high daytime activities combined with low nighttime activities. Analyses of the morphological and 16S rRNA gene diversity among cyanobacteria revealed filamentous nonheterocystous (Oscillatoriales) and unicellular (Chroococcales) representatives to be predominant. Analyses of the nifH gene diversity showed that the major phylotypes belonged to noncyanobacterial prokaryotes. However, as shown by cyanobacterial selective nifH-denaturing gradient gel electrophoresis analysis, cyanobacterial nifH gene sequences were present at all sites. Several nifH and 16S rRNA gene phylotypes were related to uncultured cyanobacteria or bacteria of geographically distant habitats, stressing the widespread occurrence of still poorly characterized microorganisms in tropical benthic marine communities.     

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.     

Antigen levels of urokinase-type plasminogen activator receptor and its gene polymorphism related to microvessel density in colorectal cancer

We determined the distribution of genotypes and frequencies of alleles of the (CA)(n) repeat polymorphism in intron 3 of the urokinase plasminogen activator receptor (uPAR) gene, uPAR antigen levels and microvessel density (MVD) in tumour and distant mucosa samples from 52 patients with colorectal cancer. The uPAR level was higher for patients with high MVD comparing to patients with lower MVD which may suggest that uPAR can be correlated with progression of colorectal cancer. The significant relationship between the high MVD and uPAR antigen level appeared to be independent of the (CA)(n) repeat polymorphism because no differences in the level of uPAR antigen between carriers of alleles were found. The received results, indicate that uPAR might be considered as a target in colorectal cancer patients' therapy.