Using environmental isotopes 2H and 18O for identification of infiltration processes in floodplain ecosystems of the River Elbe.

We examined a floodplain area in the middle section of the river Elbe Valley with regard to hydrogeological and hydrological processes using isotopic methods. Over two years, river water and groundwater have been analysed for temporal and spatial chemical and isotopic (delta2H and delta18O) changes. By these methods we assessed the flow dynamics of the river-groundwater infiltration system. At low and mean river stages there is a general hydraulic gradient from the higher areas at the margin of the valley towards the floodplain. During floods river water infiltrates into the adjacent aquifer not primarily through the river banks but first through surface water inflow from north to south, via depressions and gullies from the back of the floodplain. The early stage of river water infiltration is characterized by a sharp decrease in conductivity and in concentrations of SO4(2-) and Cl- in the hydraulically connected shallow aquifer. delta2H and delta18O values show a similar tendency. We observed a significant minimum in stable isotope ratios during the flood in March 1999. Using a simple mixing equation it was calculated that the groundwater in the upper, shallow aquifer consists of around 70% river water in the transition zone (well 13) during flooding.

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis.

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (vmax = 24.4 pmol/(mg min), K(m) = 64.4 microM) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.

Iron uptake and intracellular metal transfer in mycobacteria mediated by xenosiderophores.

Growth promotion was tested using M. smegmatis wild type strain, an exochelin-deficient mutant, and M. fortuitum employing a broad variety of xenosiderophores including hydroxamates, catecholates and alpha-hydroxy carboxylic acids. The experiments revealed that utilization of siderophore-bound iron is substrate specific suggesting high-affinity siderophore receptor and transport systems. Concentration-dependent uptake of a selected xenosiderophore (fericrocin) in M. smegmatis showed saturation kinetics and uptake was inhibited by respiratory poisons. In situ Mössbauer spectroscopy of ferricrocin uptake in M. smegmatis indicated rapid intracellular reductive removal of the metal excluding intracellular ferricrocin accumulation. The ultimate intracellular iron pool is represented by a compound (delta = 0.43 mm s-1, delta EQ = 1.03 mm s-1) which has also been found in many other microorganisms and does not represent a bacterioferritin, cytochrome or iron-sulfur cluster. By contrast, iron uptake via citrate-a compound exhibiting a very low complex stability constant-involves ligand exchange with mycobactin. Mycobactin has merely a transient role. The ultimate storage compound is an E. coli-type bacterioferritin, in which over 90% of cellular iron is located.

AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1.

Interleukin-1 (IL-1) -alpha and -beta are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo.

An ex vivo method for studying inflammation in cynomolgus monkeys: analysis of interleukin-1 receptor antagonist.

Nonhuman primates have been used as models for testing the role of interleukin-1 (IL-1) in inflammatory diseases, including endotoxemia. The objective of this investigation was to develop a reproducible and rapid method for in vivo evaluation of IL-1 antagonists using cynomolgus monkeys. IL-1 alone can induce many of the symptoms of endotoxemia in monkeys including fever, loss of appetite, and lethargy, however, test animals are slow to recover and may become desensitized to IL-1. We have developed an ex vivo method using whole blood for analysis of IL-1 antagonists administered in vivo to the monkeys and report here results for the naturally occurring IL-1 receptor antagonist, IL-1ra. In this procedure, animals are given an i.v. infusion of IL-1ra, and blood samples are taken preinfusion and during the infusion. The samples are incubated with or without IL-1 beta and the subsequent ex vivo induction of IL-6 determined. This allows analysis of the effects of in vivo pharmacodynamics on the efficacy of antagonists without exposing the test animals to IL-1. In this ex vivo protocol, each animal serves as its own control, eliminating from the assessment the large animal to animal variation observed with in vivo responses. By testing various doses, we estimate that 50% inhibition of IL-1 induced IL-6 can be achieved with an infusion of IL-1ra at 5 micrograms/kg/15 min. This method allows simple and efficient analysis of inhibitors and antagonists of IL-1 and, potentially, other effectors.

Fungal ferritins: the ferritin from mycelia of Absidia spinosa is a bacterioferritin.

Two distinct ferritin like iron containing proteins have been identified and isolated from the fungus Absidia spinosa; one from the spores and another from the mycelia. The mycelial protein has been purified and consists of two subunits of approx. 20 kDa. The N-terminal sequences of both subunits have been determined. The holoprotein as isolated contains approx. 750 iron atoms/molecule and exhibits a heme-like UV-Vis spectrum. Based on the heme spectrum and the high degree of sequence homology found, it has been established that the mycelial protein is a bacterioferritin. This is the first example demonstrating the presence of a bacterioferritin in a eukaryotic organism.

The mobile ferrous iron pool in Escherichia coli is bound to a phosphorylated sugar derivative.

Based on in vivo Mössbauer spectroscopy it has previously been demonstrated that the intracellular iron pool of Escherichia coli, grown in iron deficient media supplemented with siderophores as the sole iron source, is dominated by a single Fe2+ and a single Fe3+ species. We have isolated the ferrous ion species and have purified it employing native column PAGE, chromatography and ultrafiltration. The purified compound displays an Mapp of 2.2 kDa and an extremely low isoelectric point (pI) of 1.05. It is shown that this ferrous ion binding compound is neither a protein nor a nucleotide, rather it is composed mainly of phosphorylated sugar derivatives. This compound binds approximately 40% of the cytoplasmic iron. Therefore it is proposed that this oligomeric ferrous carbohydrate phosphate represents the long sought after mobile, low molecular mass iron pool.

Effects of exogenous glucagon on concentrations of glucose, fructose and insulin in plasma of sheep fetus.

Exogenous glucagon infused into the fetal sheep resulted in an increase in the concentration of glucose and insulin in fetal arterial plasma without a significant change in the concentration of fructose. Lack of any significant changes in glucagon, insulin, glucose and fructose concentrations in maternal plasma suggests that the alterations in the fetus are secondary to fetal metabolic and hormonal mechanisms rather than reflecting effects of maternal metabolism.

Effect of acute nonsurgical stress on fetal and maternal plasma glucose, glucagon and insulin concentration in sheep.

The purpose of this study was to investigate the effect of nonsurgical stress on plasma glucose, insulin and glucagon in the ewe and fetus. Plasma glucose, insulin and glucagon were measured before and after a 2 min period of verbal and physical startling of much greater magnitude than that to which we ewe is exposed during routine blood drawing. Studies were completed on 5 fed ewes, 5 fasted ewes and on 4 fetuses of fasted ewes. There were no significant differences after a startling compared to the control values. Thus, there appears no need to allow the ewe a prolonged period (more than 1-2 weeks) to become accustomed to handling by humans before chronic metabolic studies involving serum glucose, insulin and glucagon are undertaken.