Induction of Nramp2 in activated mouse macrophages is dissociated from regulation of the Nramp1, classical inflammatory genes, and genes involved in iron metabolism.

Nramp2 is a widely expressed metal-ion transporter that is involved in dietary iron absorption in the duodenum and iron uptake from transferrin in peripheral tissues. Nramp1 is a related gene involved in regulation of host pathogen interaction. Nramp2 has at least two alternatively spliced isoforms, one of which contains an iron-responsive element in its 3'-untranslated region. In this study, we investigated the regulation of both isoforms of Nramp2 in activated primary macrophages from mouse strains with wild-type (Bcg(r)) or mutant (Bcg(s)) alleles. The Nramp2-IRE and/or -nonIRE transcripts were up-regulated in all mouse strains analyzed after treatment with interferon-gamma and lipopolysaccharide. cDNA microarray analysis revealed that Nramp2 regulation is controlled discordantly from other iron-regulated genes and classical macrophage-activation genes in different mouse strains. We suggest that Nramp2 is regulated independently of known iron-responsive genes in macrophages, and its function in host defense is unrelated to Nramp1.

Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region.

Koalas are infected with two species of Chlamydia, C. pecorum and C. pneumoniae. While it is known that significant genetic diversity occurs in the C. pecorum strains infecting koalas, very little is known about the C. pneumoniae strains that infect this host. In the current study, 10 isolates of koala C. pneumoniae were analysed at four gene loci and found to be different to both the human and horse C. pneumoniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4). All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) gene. This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C. pneumoniae, human C. pneumoniae, guinea pig inclusion conjuctivitis C. psittaci and feline conjuctivitis C. psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations. This study also provides the first documented case of natural C. pneumoniae infection causing a severe and extended respiratory episode in a captive koala population. This captive episode is in contrast to most free-range observations in which koala C. pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.

Interferon-gamma and lipopolysaccharide regulate the expression of Nramp2 and increase the uptake of iron from low relative molecular mass complexes by macrophages.

The natural resistance associated macrophage protein 2 (Nramp2) is a transporter that is involved in iron (Fe) uptake from transferrin (Tf) and low molecular mass Fe complexes. Here we describe the effect of the inflammatory mediators interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) on the expression of Nramp2 mRNA and Fe uptake by cells of the macrophage lineage. After incubation of the RAW264.7 macrophage cell line with LPS there was a sevenfold increase in the expression of the 2.3 kb Nramp2 mRNA transcript when compared with the control, but little effect on the Nramp2 3.1 kb transcript. These results indicate differential regulation of the two transcripts. Treatment with LPS resulted in an increase in 59Fe uptake from 59Fe-nitrilotriacetic acid, while transferrin receptor (TfR) mRNA levels and 59Fe uptake from 59Fe-Tf were decreased. Paradoxically, at the same time, an increase in iron regulatory protein (IRP)1 RNA-binding activity was observed. Incubation with IFN-gamma (50 U.mL-1) resulted in a marked decrease in TfR mRNA levels but had no effect on Nramp2 mRNA expression. Exposure of RAW264.7 cells to both IFN-gamma and LPS resulted in a fourfold increase in the Nramp2 2.3-kb transcript and a four to fivefold decrease in the 3.1-kb transcript when compared with the control. Furthermore, there was a decrease in TfR mRNA levels despite an increase in IRP1 RNA-binding activity and a marked increase in inducible nitric oxide synthase mRNA expression. Hence, TfR and Nramp2 mRNA expression did not appear to be regulated in a concerted manner. Similar responses to those found above for RAW264.7 cells were also observed in the J774 macrophage cell line and also for primary cultures of mouse peritoneal macrophages. These results are of interest as the TfR and Nramp2 are thought to act together during Fe uptake from Tf. This is the first report to demonstrate regulation of the Nramp2 mRNA transcripts by inflammatory mediators.

Preliminary biochemical characterization of faeces from uninfected rats and rats infected with the tapeworm, Hymenolepis diminuta.

Faecal pellets were collected from uninfected rats and rats infected with Hymenolepis diminuta, and analysed for caloric value, lipids, carbohydrates, glucose, amino acids, and proteins. Faeces from infected rats contained significantly more lipid and less carbohydrate and glucose than faeces from control rats. The total free amino acid content of faeces from infected and control rats did not differ, but there were significant differences in the amounts of individual free amino acids. The data support the hypothesis that the nutrient composition of faeces from infected and control hosts differ. Beetles that serve as the intermediate host for H. diminuta may be able to detect these differences, and this may explain why beetles preferentially ingest faeces containing the tapeworm s eggs.

Quantification of benzoquinones in the flour beetles. Tribolium castaneum and Tribolium confusum.

Benzoquinones were extracted from flour beetles by rinsing them for 8 min in methanol followed by homogenization in fresh methanol. This procedure recovered > 96% of the total benzoquinones from both species, with the methanolic rinse and supernatant representing the "surface" and "total" benzoquinones. As determined by UV/VIS spectrometry and reversed-phase HPLC, the methanolic rinse contained methyl- and ethyl-1,4-benzoquinone (MBQ and EBQ), but no substituted hydroquinones, The methanolic supernatant contained MBQ and EBQ, and methyl- and ethyl-hydroquinone. When stored at -20 degrees C for 72 hr, the chemical composition of the methanolic rinse did not change, but the composition of the methanolic homogenate did change. Notably, the amounts of benzoquinones in the methanolic supernatant decreased during storage. The method described represents an improvement over those described previously in that it uses less hazardous solvents for extraction (methanol vs more non-polar solvents such as hexane) and allows for the differentiation and quantification of the surface and total benzoquinones of these beetles.

The effect of intracellular iron concentration and nitrogen monoxide on Nramp2 expression and non-transferrin-bound iron uptake.

Recent studies have demonstrated that the protein product (natural resistance associated macrophage protein 2, Nramp2) encoded by the gene Nramp2 acts as an Fe transporter involved in the uptake of Fe from transferrin (Tf) and low Mr Fe complexes. Interestingly, there are two splice variants of Nramp2, one with a putative iron-responsive element (IRE) in its 3' untranslated region (UTR) and another without. Due to the importance of Nramp2 in Fe transport, and the presence of an IRE in its 3'-UTR, we have examined the effect of Fe-deprivation, Fe-loading, and nitrogen monoxide on the expression of Nramp2 mRNA. These results were compared to the expression of transferrin receptor (TfR) mRNA which also has IREs in its 3'-UTR and is regulated by Fe and NO via the binding of iron-regulatory proteins (IRPs) to its IREs. Our experiments show that the IRE in Nramp2 mRNA does bind the IRPs in lysates from a mouse fibroblast cell line (LMTK-). Moreover, reverse transcription-PCR (RT-PCR) demonstrated that both the IRE and non-IRE-containing transcripts were present within these cells. However, there was no change in Nramp2 mRNA expression in LMTK- cells after a 20-h incubation with either the Fe chelator, desferrioxamine (DFO), the Fe donor, ferric ammonium citrate (FAC), or the NO generator, S-nitroso-N-acetylpenicillamine (SNAP). In contrast, these agents caused a marked change in the RNA-binding activity of the IRPs and the expression of TfR mRNA. In addition, both FAC and DFO caused an appropriate change in [59Fe] uptake from [59Fe]Tf, viz., an increase in Fe uptake after exposure to DFO and a decrease after treatment with FAC. As Nramp2 can transport Fe from non-Tf-bound Fe, the effect of preincubation with DFO and FAC was also examined on Fe uptake from [59Fe]nitrilotriacetate and [59Fe]citrate. However, in contrast to the results found for [59Fe]Tf, incubation with DFO and FAC did not result in appropriate regulation of Fe uptake from [59Fe]nitrilotriacetate or [59Fe]citrate. These data demonstrate that non-Tf-bound Fe uptake was not under control of the IRP-IRE system in these cells. Collectively, the results indicate that in LMTK-fibroblasts Nramp2 mRNA expression was not regulated like TfR mRNA.

Nitrogen monoxide activates iron regulatory protein 1 RNA-binding activity by two possible mechanisms: effect on the [4Fe-4S] cluster and iron mobilization from cells.

The iron-regulatory protein 1 (IRP1) regulates the expression of several molecules involved in iron (Fe) metabolism by reversibly binding to iron-responsive elements (IREs) in the untranslated regions (UTR) of particular mRNA transcripts. Several studies have indicated that nitrogen monoxide (NO) may influence IRP1 RNA-binding activity by a direct effect on the [4Fe-4S] cluster of the protein. It has also been suggested that NO may act indirectly on IRP1 by affecting the intracellular Fe pools that regulate the function of this protein [Pantopoulous et al. (1996) Mol. Cell. Biol. 16, 3781-3788]. There is also the possibility that NO may S-nitrosate sulfhydryl groups that are crucial for mRNA binding and decrease IRP1 activity by this mechanism. We have examined the effect of a variety of NO donors [e.g., S-nitroso-N-acetylpenicillamine (SNAP), spermine-NONOate (SperNO), and S-nitrosoglutathione (GSNO)] on IRP1 RNA-binding activity in both LMTK(-) fibroblast lysates and whole cells. In cell lysates, the effects of NO at increasing RNA-binding activity were only observed when cells were made Fe-replete. Under these circumstances, IRP1 contains an [4Fe-4S] cluster that was susceptible to NO. In contrast, when lysates were prepared from cells treated with the Fe chelator desferrioxamine (DFO), NO had no effect on the RNA-binding activity of IRP1. The lack of effect of NO under these conditions was probably because this protein does not have an [4Fe-4S] cluster. In contrast to the NO generators above, sodium nitroprusside (SNP) decreased IRP1 RNA binding when cells were incubated with this compound. However, SNP had no effect on IRP1 RNA-binding activity in lysates, suggesting that the decrease after incubation of cells with SNP was not due to S-nitrosation of critical sulfhydryl groups. Apart from the direct effect of NO on IRP1 in Fe-replete cells, we have shown that NO generated by SNAP, SperNO, and GSNO could also mobilize Fe from cells. When NO generation was induced in RAW 264.7 macrophages, an increase in IRP1 RNA-binding activity occurred but there was only a small increase in Fe release. Our results suggest that NO could activate IRP1 RNA-binding by two possible mechanisms: (1) its direct effect on the [4Fe-4S] cluster and (2) mobilization of (59)Fe from cells resulting in Fe depletion, which then increases IRP1 RNA-binding activity.

Hymenolepis diminuta: glucose and glycogen gradients in the adult tapeworm.

Adult (20-day-old) Hymenolepis diminuta were cut into 12 pieces of equal length, and the individual pieces of the tapeworm's strobila were analyzed. There was a continuous gradient of decreasing concentrations of glucose (mM) and decreasing levels of glycogen (microgram/mg wt) in the strobila. The ethanol extracts of the individual pieces of strobila contained four compounds tentatively identified as disaccharides; the distributions of these compounds were different from those of glucose and glycogen. The distributions of glucose, glycogen, and the "disaccharides" changed when tapeworms were incubated for 1 h in saline or glucose. When compared, on a per-weight basis, to the most posterior sections of the strobila, the anterior sections absorbed more glucose and incorporated more glucose into glycogen. There was a continuous gradient of decreasing values along the tapeworm's strobila of the Vmax for glucose uptake, while the Kt values for glucose uptake changed only slightly. The data indicate that glucose and glycogen metabolism are most active in the anterior part of the tapeworm's strobila where new proglottids are produced and the initial stages of organogenesis occur.

Effect of brood size manipulation on offspring physiology: an experiment with passerine birds.

The environment experienced during ontogeny has a significant impact on the physiological condition of offspring. This, in turn, forecasts survival probabilities and future reproductive potential. Despite the prominent role that the concept of condition plays in evolutionary studies, the physiological and biochemical characters that define it remain relatively unexplored. In this study, we quantified the impact of brood size manipulations on the physiology and biochemistry of nestling tree swallows (Tachycineta bicolor) shortly before they fledged. Over two breeding seasons, we either increased or decreased the number of individuals in a brood by a single nestling. Every 2-4 days, we determined the resting rate of oxygen consumption [V(O(2))] of individuals in each brood. Growth was followed until 16 days of age, at which time, to look for potential trade-offs in energy allocation, we measured total lipid mass, skeletal muscle and organ mass, indices of blood oxygen-carrying capacity and the activities of key metabolic enzymes in various tissues. Surprisingly, there was a minimal response of most characters to brood manipulation, suggesting that physiological and biochemical development is relatively invariant except perhaps under extreme conditions. Individuals reared in artificially enlarged broods, however, had a significantly lower body mass, body-size-adjusted [V(O(2))], gizzard mass and total lipid mass. These individuals also had decreased activity of cardiac 3-hydroxyacyl CoA dehydrogenase, suggesting a decreased capacity for oxidation of fatty acids. How these characters affect survival or the future adult phenotype remains unknown.