Modulation of granulocyte functions by peptide YY in the rat: Age-related differences in Y receptors expression and plasma dipeptidyl peptidase 4 activity

It has been acknowledged that aging exerts detrimental effects on cells of the innate immune system and that neuropeptides, including neuropeptide Y (NPY) and NPY-related peptides fine-tune the activity of these cells through a receptor specific mechanism. The present study investigated the age-dependent potential of peptide YY (PYY) to modulate different granulocyte functions. The PYY reduced the carrageenan-elicited granulocyte accumulation into the air-pouch of aged (24 months) rats, and markedly decreased the phagocytosis of zymosan, as well as the H₂O₂ production, when applied in vivo (20 μg/air-pouch). The anti-inflammatory effect of PYY was less prominent in adult (8 months) and young (3 months) rats. However, the proportions of granulocytes expressing Y1, Y2 and Y5 receptor subtypes were significantly lower in both aged and young rats when compared to adult rats. Furthermore, the aging was found to be associated with the diminished dipeptidyl peptidase 4 (DP4, an enzyme converting the NPY and PYY to Y2/Y5 receptor selective agonists) activity in plasma. In conclusion, the diverse age-related anti-inflammatory effect of PYY in rats originates from different expression levels of Y1, Y2, and Y5 receptor subtypes in addition to different plasma DP4 activity.     

Basidiomycetes harbour a hidden treasure of proteolytic diversity

This survey is the first to investigate the proteolytic potential of a large number of basidiomycetes. Aqueous extracts of 43 basidiomycetes were investigated for their content of proteolytic activities, using gelatin zymography. The activities were characterised qualitatively using class specific inhibitors. All four catalytic classes of proteases were present, with 4% of all activities classified as aspartic, 5% as cysteine, 6% as metallo and 22% as serine proteases, while the remaining activities could not be assigned unambiguously. The majority of the latter were not inhibited by any of the inhibitors used and were termed insensitive. Different proteolytic activities are evenly distributed among members of all orders of basidiomycetes, although some taxa are a richer source of proteases than others. A significant number of the cysteine protease activities shown here have not previously been reported in basidiomycetes. The fungal cysteine and serine protease inhibitors, clitocypin and CNSPI (Clitocybe nebularis serine protease inhibitor), both inhibited a number of activities and even a few activities that were otherwise insensitive to all other inhibitors used, hence indicating their potential for a regulatory role. The number and diversity of proteases in basidiomycetes are seen to be remarkable and encourage further investigation.     

Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions

Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.     

Intensity of mineralization in the hyporheic zone of the prealpine river Bača (West Slovenia)

Respiratory electron transport system (ETS) activity and oxygen consumption in the interstitial water, and in the fine (i.e. silt) and coarse (sand) sediment fractions from the hyporheic zone of the prealpine river Bača (W Slovenia) have been measured in order to estimate the intensity of potential and actual carbon mineralization through microbial communities. Hyporheic samples from the river bed (RB) and gravel bars (GB) were compared. ETS activity and oxygen consumption of all fractions from the RB did not differ significantly from those from the GB. ETS activity and oxygen consumption of biofilm attached to 1 g of the silt were higher than of that attached to the same mass of the sand. A significant correlation between ETS activity and oxygen consumption indicated that the former should be a good indicator of intensity of bioactivity in hyporheic sediments. The ratio of ETS activity to oxygen consumption (ETS/R ratio) revealed that the oxygen consumption of microorganisms is responsible for approximately 60% of the metabolic potential in the hyporheic sediments. The contributions of different fractions of sediment to the total ETS activity differed between RB and GB. The contribution of microorganisms in the interstitial water and silt was higher in GB than in the RB, but the sand fraction contributed less to potential carbon loss in GB than in the RB. Average total respiratory carbon loss per volume through the hyporheic zone was higher in the RB than in GB. The main reasons suggested are the different intensity of exchange of surface water with the hyporheic zone, and the rate of consolidation of sediments, which is primarily a function of river hydrology and geomorphology. Handling editor: J. Padisak     

Function of a Novel Cadmium-Induced YodA Protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cells of Escherichia coli increase greatly the synthesis of a small primarily cytoplasmic protein as soon as the cell growth rate falls below the maximal growth rate supported by cadmium exposure, after which the mature product is exported to the periplasm. This protein was previously identified as the product of the E. coli yodA open reading frame. We now report the isolation of an E. coli mutant defective in YodA synthesis because of insertional inactivation of the corresponding gene. In experiments to test the ability of both the wild-type and yodA mutant E. coli cells to bind cadmium, we have used γ-labeled [¹⁰⁹Cd]. Whereas the wild-type E. coli strain was able to bind metal, the yodA mutant strain failed to do so. In addition, analysis of such a mutant demonstrated that it grows at a rate distinguishable from that of the isogenic parent in the presence of cadmium ions. However, challenging cells with hydrogen peroxide and additional metals such as zinc, copper, cobalt, and nickel did not significantly affect the growth rate of the mutant. This growth phenotype was found to be the result of the loss of its ability to bind cadmium. These results suggest that the role of YodA protein might be to decrease the concentration level of cadmium ions in E. coli cells during cadmium stress by its ability to bind heavy metal.     

Emergence of uropathogenic extended-spectrum beta lactamases-producing Escherichia coli strains in the community

The aim of this study was to determine the virulence characteristics and resistance pattern of the extended-spectrum/lactamases (ESBLs)-producing Escherichia coli strains isolated from urine of outpatients in the Zagreb region during a five-month period, and to compare them with the non ESBLs-producing E. coli strains isolated in the same period. Out of 2451 E. coli strains isolated from urine of nonhospitalized patients with significant bacteriuria, a total of 39 ESBLs-producing strains (1.59%) were detected by a double-disk diffusion technique and by the broth-dilution minimal inhibitory concentration reduction method. The 45 non ESBLs-producing strains were randomly chosen, and phenotype of the two groups of strains was characterized and compared. Serogroup O4, hemolysin production, expression of P- and type 1 fimbriae as well as resistance to gentamicin and amikacin were significantly more prevalent characteristics among the ESBLs-producing strains than among non ESBLs-producing strains (p < 0.01), while higher prevalence of trimethoprim-sulfamethoxazole resistance among ESBLs-producing strains was not statistically significant (p > 0.05). Chromosomal DNA analysis by pulsed-field gel electrophoresis exhibited a great genomic similarity among ESBLs-producing strains and revealed that those highly virulent and resistant E. coli strains isolated from urine of outpatients in the Zagreb region had a clonal propagation.     

Identification of six putative human transporters with structural similarity to the drug transporter SLC22 family

The solute carrier family 22 (SLC22) is a large family of organic cation and anion transporters. These are transmembrane proteins expressed predominantly in kidneys and liver and mediate the uptake and excretion of environmental toxins, endogenous substances, and drugs from the body. Through a comprehensive database search we identified six human proteins not yet cloned or annotated in the reference sequence databases. Five of these belong to the SLC22 family, SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, and the sixth gene, SVOPL, is a paralog to the synaptic vesicle protein SVOP. We identified the orthologs for these genes in mouse and rat and additional homologous proteins and performed the first phylogenetic analysis on the entire SLC22 family in human, mouse, and rat. In addition, we performed a phylogenetic analysis which showed that SVOP and SV2A-C are, in a comparison with all vertebrate proteins, most similar to the SLC22 family. Finally, we performed a tissue localization study on 15 genes on a panel of 30 rat tissues using quantitative real-time polymerase chain reaction.     

The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test

The most commonly used toxicity test worldwide is the acute Daphnia magna test. The relevance of acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity in D. magna exposed to chromium, cadmium, and diazinon was evaluated in connection with this standard test. We found no link between enzyme activities and immobility. Concentrations of Cr(6+) up to 280 microg/L had no effect on AChE and GST activities, while 20% immobility was observed. At concentrations of 20-25 microg/L of Cd(2+) AChE activity was increased by about 50%. The effect of diazinon on both enzymes was insignificant up to concentrations that caused 27% immobility. Consequently, while the use of AChE and GST activities is recommended when the mode of action of chemicals is studied, the value of these biomarkers in routine acute toxicity tests is limited because the relationship between enzyme activities and immobility of D. magna exposed to different chemicals is unclear.