Pericryptal Myofibroblast Growth in Rat Descending Colon Induced by Low-Sodium Diets Is Mediated by Aldosterone and not by Angiotensin II

Pericryptal myofibroblast growth in descending colonic crypts correlates with the activation of the renin-angiotensin-aldosterone system. Earlier work showed that during the transition from a high-Na⁺ (HS) to low-Na⁺ (LS) diet there are changes in the colonic crypt wall and pericryptal sheath. As LS diet increases both aldosterone and angiotensin II, the aim here was to determine their individual contributions to the trophic changes in colonic crypts. Experiments were conducted on control and adrenalectomized Sprague-Dawley rats fed an HS diet and then switched to LS diet for 3 days and supplemented with aldosterone or angiotensin II. The actions of the angiotensin-converting enzyme inhibitor captopril, the angiotensin receptor antagonist losartan and the aldosterone antagonist spironolactone on extracellular matrix proteins, claudin 4 and E-cadherin myofibroblast proteins, α-smooth muscle actin (α-SMA) and OB-cadherin (cadherin 11), angiotensin type 1 and TGFβr1 membrane receptors were determined by immunolocalization in fixed distal colonic mucosa. The LS diet or aldosterone supplementation following ADX in HS or LS increased extracellular matrix, membrane receptors and myofibroblast proteins, but angiotensin alone had no trophic effect on α-SMA. These results show that aldosterone stimulates myofibroblast growth in the distal colon independently of dietary Na⁺ intake and of angiotensin levels. This stimulus could be a genomic response or secondary to stretch of the pericryptal sheath myofibroblasts accompanying enhanced rates of crypt fluid absorption.     

Intracellular trafficking of CD23: differential regulation in humans and mice by both extracellular and intracellular exons

In mouse models of food allergy, we recently characterized a new CD23b-derived splice form lacking extracellular exon 5, bDelta5, which undergoes constitutive internalization and mediates the transepithelial transport of free IgE, whereas classical CD23b is more efficient in transporting IgE/allergen complexes. These data suggested that regulation of endocytosis plays a central role in CD23 functions and drove us to systematically compare the intracellular trafficking properties of human and murine CD23 splice forms. We found that CD23 species show similar endocytic behaviors in both species; CD23a undergoes constitutive clathrin-dependent internalization, whereas CD23b is stable at the plasma membrane. However, the mechanisms controlling these similar behaviors appeared to be different. In mice, a positive internalization signal was localized in the cytoplasmic region shared by all CD23 splice forms. This positive signal was negatively regulated by the intracellular CD23b-specific exon. In addition, the fact that alternative splice forms lacking exons of the extracellular region (5, 6, 7, and/or 8) were all constitutively internalized suggested that endocytosis of murine CD23 is regulated by a process similar to the outside-in signaling of integrins. In humans, the internalization signal was mapped in the CD23a-specific intracellular exon. Interestingly, this signal also behaved as a basolateral targeting signal in polarized Madin-Darby canine kidney cells. The latter result and the fact that human intestinal cell lines were found to coexpress both CD23a and CD23b provide a molecular explanation for the initial observations that CD23 was found at the basolateral membrane of intestinal epithelial cells from allergic patients.     

Drosophila alcohol dehydrogenase: acetate-enzyme interactions and novel insights into the effects of electrostatics on catalysis

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones and that is also able to further oxidize aldehydes to their corresponding carboxylic acids. The structure of the ternary enzyme-NADH-acetate complex of the slow alleloform of Drosophila melanogaster ADH (DmADH-S) was solved at 1.6 A resolution by X-ray crystallography. The coenzyme stereochemistry of the aldehyde dismutation reaction showed that the obtained enzyme-NADH-acetate complex reflects a productive ternary complex although no enzymatic reaction occurs. The stereochemistry of the acetate binding in the bifurcated substrate-binding site, along with previous stereochemical studies of aldehyde reduction and alcohol oxidation shows that the methyl group of the aldehyde in the reduction reaction binds to the R1 and in the oxidation reaction to the R2 sub-site. NMR studies along with previous kinetic studies show that the formed acetaldehyde intermediate in the oxidation of ethanol to acetate leaves the substrate site prior to the reduced coenzyme, and then binds to the newly formed enzyme-NAD+ complex. Here, we compare the three-dimensional structure of D.melanogaster ADH-S and a previous theoretically built model, evaluate the differences with the crystal structures of five Drosophila lebanonensis ADHs in numerous complexed forms that explain the substrate specificity as well as subtle kinetic differences between these two enzymes based on their crystal structures. We also re-examine the electrostatic influence of charged residues on the surface of the protein on the catalytic efficiency of the enzyme.     

Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells

We compared the intracellular distribution and regulatory role of fatty acid transporter protein (FATP1) and fatty acid translocase (FAT/CD36) on muscle cell fatty acid metabolism. With the use of adenoviruses, FATP1 and FAT genes were delivered to primary cultured human muscle cells. FATP1 and FAT moderately enhanced palmitate and oleate transport evenly at concentrations of 0.05, 0.5, and 1 mM. Long-term (16 h) consumption of palmitate and oleate from the media, and particularly incorporation into triacylglyceride (TAG), was stimulated equivalently by FATP1 and FAT at all fatty acid concentrations tested. In contrast, long-term CO₂ production was reduced by FATP1 and FAT at all doses of palmitate and at the lower concentrations of oleate. Neither FATP1 nor FAT markedly altered the production of acid-soluble metabolic intermediates from palmitate or oleate. The intracellular localization of fusion constructs of FATP1 and FAT with enhanced green fluorescent protein (EGFP) was examined. Independently of fatty acid treatment, FATPGFP was observed throughout the cytosol in a reticular pattern and concentrated in the perinuclear region, partly overlapping with the Golgi marker GM-130. FATGFP was found in the extracellular membrane and in cytosolic vesicles not coincident with GM-130. Neither FATP1 nor FAT proteins colocalized with lipid droplets in oleate-treated cells. We conclude that whereas FAT is localized on the extracellular membrane, FATP1 is active in the cytosol and imports fatty acids into myotubes. Overall, both FATP1 and FAT stimulated transport and consumption of palmitate and oleate, which they channeled away from complete oxidation and toward TAG synthesis. palmitate; oleate; fatty acid binding proteins; skeletal muscle     

Sex-related differences in energy balance in response to caloric restriction

Sex-related differences in energy balance were studied in young Wistar rats fed standard chow pellets either ad libitum or in restricted amounts (60% of ad libitum intake) for 100 days. Caloric intake, indirect calorimetry, organ and adipose tissue weights, energy efficiency, liver mitochondrial respiration rate, and brown adipose tissue (BAT) uncoupling protein-1 (UCP1) content were measured. Ad libitum-fed females showed greater oxygen consumption (Vo(2)) and carbon dioxide production (Vco(2)) and lower energy efficiency than males. Caloric restriction induced a chronic drop of Vo(2) and Vco(2) in females but not in males over the period studied. Restricted females showed a better conservation of metabolic active organ mass and a greater decrease in adipose depots than restricted males. Moreover, changes of BAT size and UCP1 content suggest that BAT may be the main cause responsible for sex differences in the response of energy balance to caloric restriction. In conclusion, our results indicate that females under caloric restriction conditions deactivate facultative thermogenesis to a greater degree than males. This ability may have obvious advantages for female survival and therefore the survival of the species when food is limiting.     

Epidemiology of Cryptosporidium molnari in Spanish gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) cultures: from hatchery to market size

A long-term epidemiological study of Cryptosporidium molnari in aquacultured European sea bass (ESB) and gilthead sea bream (GSB) was performed in different types of facilities on the Atlantic, Cantabric, and Mediterranean coasts. Four types of studies were carried out. In study A, fish raised from juveniles to marketable size (ongrowing stage) were periodically sampled in three different types of cultures. Studies B and C focused on hatchery and nursery facilities. In study D, occasional samplings were performed during mortality or morbidity outbreaks. As a general trend, C. molnari was more prevalent in GSB than in ESB. Data on the distribution pattern of C. molnari in total sampled GSB (studies A, B, and D) had a variance higher than the mean (overdispersion). In GSB (study A), the type of ongrowing system (sea cages, earth ponds, or indoor tanks) was found to have no significant effect. There was a significant relationship between the presence of the parasite and both fish weight and season. The highest infection values were recorded in spring. Prevalence and intensity had convex weight profiles, with a peak in 30- to 100-g fish. In study D, the prevalence of infection was higher in fish recently introduced in sea cages and in preongrowing systems. In studies B and C, fish were almost never infected before entering the postlarval and nursery facilities. The parasite seems to enter the host mainly through the water in production steps with less stringent water treatment. Recirculation systems and fish cannibalism could contribute to oocyst concentration and dispersion in aquaculture facilities.     

Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1-receptor selective antagonists pirenzepine (10 microM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 microM) and AF-DX 116 (10 microM). The M4-receptor selective antagonists tropicamide (1 microM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 microM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals.     

Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali

The Fulani are known to be less susceptible to Plasmodium falciparum malaria infections and to have lower parasitaemia despite living under similar malaria transmission intensity compared with other ethnic tribes. The aim of the present study was to examine whether the Fulani were more polarised towards Th2 as reflected by higher numbers of malaria-specific IL-4- and IL-10-producing cells and lower numbers of IFN-gamma- and IL-12-producing cells as compared to their neighbour ethnic tribe, the Dogon of Mali. Total IgE and both anti-malaria IgE and IgG antibodies were measured by ELISA and the numbers of IL-4-, IFN-gamma-, IL-10- and IL-12-producing cells were enumerated using enzyme-linked ImmunoSpot assay (ELISPOT). Numbers of parasite clones were detected by polymerase chain reaction (PCR). The study was performed outside the transmission period and all individuals included were asymptomatic. The results revealed that the Fulani were less parasitised, had fewer circulating parasite clones in their blood, had significantly higher anti-malaria IgG and IgE antibodies and higher proportions of malaria-specific IL-4- and IFN-gamma-producing cells compared to the Dogon. The higher antigen-specific production of IL-4 among the Fulani was statistically significant both before and after adjustment for level of spontaneous cytokine production, while greater IFN-gamma production only attained statistical significance after adjustment for spontaneous levels. Taken together, the association of higher anti-malarial IgE and IgG antibodies and increased numbers of specific IL-4- and IFN-gamma-producing cells compared to the ethnic sympatric tribe, the Dogon, may assist in explaining the lower susceptibility to malaria observed in the Fulani.