Endocytosis via galactose receptors in vivo : Ligand size directs uptake by hepatocytes and/or liver macrophages

We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.     

Dietary intake of platinum and gold by children from Germany using duplicate portion sampling

The dietary intake of platinum and gold by 84 small children, 42 boys and 42 girls at the age of 14 to 83 months, with different food consumption behaviour living in urban and rural areas of Germany was measured by the duplicate method with a seven day sampling period from May to September 1998. The levels in the food duplicates were in the range of < 0.01 to 450 ng Pt/kg (dry weight) (median: 22) and < 0.14 to 28 microg Au/kg (dry weight) (median: 0.645). Related to the body weight, Pt was in the range of < 0.81 to 32 ng/(kg (body weight) x week) (median: 2.3) and Au was < 0.015 to 2.6 microg/(kg (body weight) x week) (median: 0.068). Children consuming exclusively products from the super market showed slightly higher Pt concentrations in the food duplicates and a higher dietary intake per body weight than children with food consumption including products from the family owned vegetable gardens or the surrounding area and/or products from domestic animals of the surrounding area. No influence of the food consumption behaviour was found for the concentrations in the food duplicates or the dietary intake of Au.     

Benzoate-driven dehalogenation of chlorinated ethenes in microbial cultures from a contaminated aquifer

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene (cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to-cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immunity of Fetal Mice to Prenatal Administration of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: Subcutaneous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) HC1 (25 mg/kg) in pregnant female mice at the 17th day of gestation markedly depleted striatal dopamine (DA) concentrations in the mothers 24 h later and at 24 h and 28 days after delivery. By contrast, in the offspring of the female mice exposed to MPTP during pregnancy, fetal brain DA concentrations at 24 h after injection and at 24 h after birth and striatal DA levels at 14 and 28 days postnatally were unaffected and identical to those in age-matched controls. The postnatal ontogenesis of striatal DA levels was identical in offspring of control vehicle- and MPTP-treated pregnant mice. Also, prenatal challenge with MPTP did not make nigrostriatal DA neurons more vulnerable to a second postnatal treatment with the toxin. Striatal DA depletions were identical in 6-week-old mice given MPTP, whether they were exposed to MPTP or to vehicle in utero. Monoamine oxidase (EC 1.4.3.4; MAO) type B activity was extremely low in the fetal brain and, relatively, much lower than that of MAO-A. Prenatal MPTP administration reduced maternal striatal and also embryonal brain MAO-B activity at 24 h post treatment but did not alter the normal postnatal development of striatal MAO-A and -B activities in the offspring. Study suggests that resistance of fetal DA neurons to the DA-depleting effect of MPTP may be due, at least in part, to an absence in the embryonal brain of adequately developed MAO-B activity required for the conversion of MPTP to its toxic metabolite, 1-methyl-4-phenylpyridinium ion.     

Auxin-conjugate hydrolysis in Chinese cabbage: Characterization of an amidohydrolase and its role during infection with clubroot disease

Auxin conjugates play a role in the regulation of free indole-3-acetic acid (IAA) content in plants. Not much is known about the enzymes involved in either conjugate synthesis or hydrolysis. In this study we have isolated and characterized an auxin conjugate hydrolase from Chinese cabbage seedlings and investigated it during the development of both the Chinese cabbage plants and the clubroot disease. The hydrolase isolated from light- and dark-grown seedlings accepted the amide conjugates indole-3-acetic acid-alanine (IAAla), IAA-phenylalanine (IAPhe), but not IAA-aspartate (IAAsp) as substrates. We also found a substantial amount of hydrolysis of an ester conjugate (IAA-glucose, IAGlu) in our enzyme preparation. The tentative reaction product IAA was identified by HPLC and subsequent GC-MS analysis. The pH optima for the different substrates were not identical, suggesting several hydrolase isoforms. After gel filtration chromatography we found at least two peaks containing different hydrolase isoforms. The isoform, which converted IAGlu to IAA, exhibited a molecular mass of ca 63 kDa, and an isoform of ca 21 kDa converted IAAla and IAPhe. The increased free IAA content in clubroot-diseased roots of Brassicaceae can be due to either de novo synthesis or release of IAA from conjugates. To answer this question free, ester- and amide-bound IAA was measured in 24- and 30-day-old leaves and roots of healthy and Plasmodiophora brassicae-infected Chinese cabbage, and the hydrolase activity with different substrates measured in the same tissues. The amide conjugates were dramatically enhanced in infected roots, whereas free IAA was only slightly enhanced compared to the control tissue. Hydrolase activity was also enhanced in clubbed roots, but the substrate specificity differed from that found in the seedlings. Especially, IAAsp hydrolysis was induced after inoculation with P. brassicae. We conclude that different auxin conjugates can be hydrolyzed at different developmental stages or under stress.     

Temporal dynamics of the carbon isotope composition in a Pinus sylvestris stand: from newly assimilated organic carbon to respired carbon dioxide

The (13)C isotopic signature (C stable isotope ratio; delta(13)C) of CO(2) respired from forest ecosystems and their particular compartments are known to be influenced by temporal changes in environmental conditions affecting C isotope fractionation during photosynthesis. Whereas most studies have assessed temporal variation in delta(13)C of ecosystem-respired CO(2) on a day-to-day scale, not much information is available on its diel dynamics. We investigated environmental and physiological controls over potential temporal changes in delta(13)C of respired CO(2) by following the short-term dynamics of the (13)C signature from newly assimilated organic matter pools in the needles, via phloem-transported organic matter in twigs and trunks, to trunk-, soil- and ecosystem-respired CO(2). We found a strong 24-h periodicity in delta(13)C of organic matter in leaf and twig phloem sap, which was strongly dampened as carbohydrates were transported down the trunk. Periodicity reappeared in the delta(13)C of trunk-respired CO(2), which seemed to originate from apparent respiratory fractionation rather than from changes in delta(13)C of the organic substrate. The diel patterns of delta(13)C in soil-respired CO(2) are partly explained by soil temperature and moisture and are probably due to changes in the relative contribution of heterotrophic and autotrophic CO(2) fluxes to total soil efflux in response to environmental conditions. Our study shows that direct relations between delta(13)C of recent assimilates and respired CO(2) may not be present on a diel time scale, and other factors lead to short-term variations in delta(13)C of ecosystem-emitted CO(2). On the one hand, these variations complicate ecosystem CO(2) flux partitioning, but on the other hand they provide new insights into metabolic processes underlying respiratory CO(2) emission.     

Delayed oviposition: a female strategy to counter infanticide by males?

Conflicts of interest between males and females can lead toan evolutionary arms race in which adaptations of each sex coevolve.Intersexual conflict is extreme in the brood caring, semelparousspider Stegodyphus lineatus; males encountering females thathave already produced their usually single egg sac attempt toremove and discard the egg sac and then remate with the female. Femalesthat cannot defend their eggs lose valuable time and fecundityby having to replace the clutch. Selection should favor femalesthat complete their suicidal maternal care as quickly as possiblebecause of the high risk of predation. However, some femalestake up to four times longer to oviposit than others. I proposethat females minimize the risk of male infanticide by postponingoviposition. Accordingly, early-maturing females, who sufferthe highest risk of infanticide by males, should have a longerinterval between maturation and oviposition than late-maturingfemales. The date of maturation significantly predicted theinterval between maturation and oviposition and explained upto 35% of its variation in a data set from a natural population andlonger term data from a seminatural, enclosed population. Bodysize was predicted to have a weak effect on the timing of ovipositionand was consistently less important than maturation date. Theobserved facultative timing of oviposition may have evolvedas a result of intersexual conflict over mating.