Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.     

Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Abstract: While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.     

Conversion of TNF alpha from antiproliferative to proliferative ligand in mouse intestinal epithelial cells by regulating mitogen-activated protein kinase.

The mechanisms regulating the balance between intestinal epithelial cell proliferation and differentiation are essential to maintaining an intact mucosal barrier. Mitogen-activated protein (MAP) kinases appear to be key transducers of extracellular signals in these pathways. The goal of this study was to investigate the regulation of MAP kinase by tumor necrosis factor alpha (TNFalpha) and epidermal growth factor (EGF) in intestinal epithelial cells. The young adult mouse colon cell line was studied for TNFalpha and/or EGF regulation of MAP kinase in the presence or absence of the MAP kinase kinase (MEK1) inhibitor PD 98059. Proliferation was determined by hemocytometry, and activated MAP kinase was identified by Western blot analysis, in vitro kinase assay, and confocal laser immunofluorescent microscopy. TNFalpha stimulated sustained nuclear MAP kinase activity, while EGF stimulated transient cytoplasmic MAP kinase activity. Changing TNFalpha's sustained MAP kinase activation to transient converted TNFalpha from an anti-proliferative to a proliferative ligand. These findings demonstrate that both TNFalpha and EGF activate MAP kinase in intestinal epithelial cells. The kinetics and subcellular distribution of this enzyme activity may be pivotal in the transduction of divergent cellular responses in the intestinal epithelium with implications for altered proliferative signals in inflammatory bowel disease.     

The Chaetopterus vitelline envelope is not necessary for the gamete interactions that lead to fertilization

It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.     

Ecomorphology ofCrassostrea cucullata (Born, 1778) (Ostreidae) in a mangrove creek (Gazi,Kenya)

Variations of shell form and shell length were studied for oysters growing in the mangroves of Gazi Creek, Kenya, and related to different environmental factors.For the study of the form, Fourier analysis was performed on the circumference of 85 oysters. The resulting coefficients were compared among specimens using cluster analysis. The correspondence between this classification and substrate diameter is virtually perfect (only one misclassification out of 85 oysters). For the clusters based on height above chart datum, 9 specimens, all on intermediate height levels, were misclassified. Orientation with respect to tidal current had 13 misclassifications. Mangrove species seemed to influence form only marginally, if at all.In the study of the size ofCrassostrea cucullata, the length of 956 oysters, growing along two transects were measured, and correlated with several environmental factors. Oyster length was not related to substrate diameter or its orientation with respect to the main current. Length was not influenced by density up to a cover of 70%. For densities higher than 70%, there was a fairly strong negative correlation (r ² = 0.634,n = 217). Length was not correlated with height above bottom (base of the tree) for heights lower than 20 cm, while oysters growing closer to the bottom were smaller. The correlation with height above chart datum was negative but very low (r ² = 0.060,n = 957). However, if all measurements of oysters closer than 20 cm to the bottom, and all from a density of more than 70% cover are deleted from the data set, the correlation with height increased dramatically, the slope still being negative (r ² = 0.859,n = 543).     

Assay for cellobiose dehydrogenase in the presence of laccase

The currently used assay for cellobiose dehydrogenase (CDH), an enzyme produced by many wood degrading fungi, lacks specificity and can give false results. The presence of laccase interferes with the standard assay. We have developed an assay for CDH that is insensitive to the presence of both laccase and other phenoloxidases. The method is based on the decrease of reducing end groups in lactose determined by the DNS method. Ferricyanide is present as electron acceptor. Advantages and drawbacks of CDH assay methods are discussed     

Feeding of Acartia tonsa Dana (Copepoda,Calanoida): predation on nauplii of Canuella perplexa T. et A. Scott (Copepoda,Harpacticoida) in the sluice-dock at Ostend

Feeding experiments in which the zooplankton fraction of the Sluice-dock smaller than 200 μm is offered to Acartia tonsa, the dominant calanoid of this biotope, show that it feeds on the nauplii of Canuella perplexa, the dominant benthic harpacticoid. The ecological implications of this carnivorous feeding, occurring in the presence of natural phytoplankton concentrations, are briefly discussed.     

Aminosalicylic acid inhibits IkappaB kinase alpha phosphorylation of IkappaBalpha in mouse intestinal epithelial cells

Tumor necrosis factor alpha (TNFalpha)-stimulated nuclear factor (NF) kappaB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFkappaB inhibitory protein (IkappaB) leading to its degradation and NFkappaB activation, is regulated by the multimeric IkappaB kinase complex, including IKKalpha and IKKbeta. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFalpha-regulated IkappaB degradation and NFkappaB activation. To determine the mechanism of 5-ASA inhibition of IkappaB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFalpha-stimulated phosphorylation of IkappaBalpha in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IkappaBalpha fusion protein by cellular extracts or immunoprecipitated IKKalpha isolated from cells treated with TNFalpha is inhibited by 5-ASA. Recombinant IKKalpha and IKKbeta autophosphorylation and their phosphorylation of glutathione S-transferase-IkappaBalpha are inhibited by 5-ASA. However, IKKalpha serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKalpha is predominantly responsible for IkappaBalpha phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFalpha-stimulated IKKalpha kinase activity toward IkappaBalpha in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFalpha activation of NFkappaB.     

Endocytosis of heparin-binding protein (CAP37) is essential for the enhancement of lipopolysaccharide-induced TNF-alpha production in human monocytes

Heparin-binding protein (HBP), also known as CAP37, is a proteolytically inactive serine protease homologue that is released from activated granulocytes. However, HBP is not a biologically inactive molecule but rather a multifunctional protein with properties that include the enhancement of LPS-induced TNF-alpha production from monocytes. We have previously demonstrated that HBP is internalized in monocytes. In the current study, we hypothesize that HBP is internalized in monocytes via endocytosis, and this internalization is an important mechanism by which HBP enhances LPS-induced TNF-alpha release. Using whole blood from healthy donors and flow cytometry, we found that colchicine (0.1-10 mM), cytochalasin D (1000 microM), NH4Cl (10-50 mM), and bafilomycin A1 (0.1-3 microM) significantly reduced the affinity of FITC-HBP for CD14-positive monocytes. Using isolated human monocytes and ELISA, we found that colchicine (0.1 mM), cytochalasin D (30 and 300 microM), NH4Cl (30 mM), and bafilomycin A1 (1 microM) significantly reduced the effect of HBP (10 microg/ml) to enhance LPS (10 ng/ml)-induced TNF-alpha release after 24 h. These findings demonstrate that internalization of HBP in monocytes is essential for the enhancement of LPS-induced TNF-alpha release. Transport of HBP to an activating compartment depends on intact F-actin polymerization and endosomal acidification, an important mechanism for endosomal protein sorting and trafficking.     

Epidermal and hepatocyte growth factors stimulate chemotaxis in an intestinal epithelial cell line

The migration of intestinalcells is important in the development and maintenance of normalepithelium, in a process that may be regulated by growth factors andcytokines. Although a number of growth factor receptors are expressedby intestinal cells, little progress has been made toward assignment offunctional roles for these ligand-receptor systems. This study comparesseveral growth factors and cytokines for their chemoattraction of the mouse small intestinal epithelial cell line. Epidermal and hepatocyte growth factors stimulated a rapid 30-fold chemotaxis of cells withdelayed threefold migration toward transforming growth factor-1. Despite stimulating proliferation, keratinocyte, fibroblast, or insulin-like growth factors did not stimulate directed migration. Chemotaxis required tyrosine kinase and phosphatidylinositolphospholipase C activities but not protein kinase C ormitogen-activated protein kinase activity. These findings suggest thatthe repertoire of growth factors capable of regulating directedintestinal epithelial cell migration is limited and that a divergenceexists in the signal transduction pathways for directed vs. nondirected migration.