Isolation and biological characteristics of chicken adipose-derived progenitor cells

Adipose-derived stem cells/adipose-derived progenitor cells (ADPCs) are multipotent stem cells that can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit, and other mammals but rarely from poultry. In this study, ADPCs were isolated from 1-day-old chicks. Primary ADPCs were subcultured to passage 15. The surface markers of ADPCs, CD29, CD44, CD71, and CD73, were detected by immunofluorescence and RT-polymerase chain reaction assays. The growth curves of different passages were all typically sigmoidal. In addition, ADPCs of different passages were successfully induced to differentiate into osteoblasts, adipocytes, and myocardial cells. The results suggest that the ADPCs isolated from chicken possess similar biological characteristics with those derived from other species, and their multilineage differentiation provides many potential applications.     

Toxicity of methylglyoxal towards rat enterocytes and colonocytes.

Effect of methylglyoxal, a bacterial metabolic product, on protein, DNA, and RNA synthesis in rat enterocytes and colonocytes was investigated. Results showed that 1 mM methylglyoxal inhibited protein, DNA, and RNA synthesis to the extent of 65-85, 65-80, and 10-20 per cent, respectively, in villus and crypt cells and colonocytes. The inhibitory pattern was similar in these various cell types. The inhibitory effect on protein and DNA synthesis was more marked than that on RNA synthesis. Inclusion of thiol compounds up to 4 mM concentration did not protect the cells from the inhibitory effect of methylglyoxal. No alteration in the level of cellular reduced glutathione and glyoxalase enzyme activity was observed when cells were incubated with 2 mM methylglyoxal. These results suggest that the antiproliferative action of methylglyoxal on eukaryotic cells may be through the inhibition of macromolecular synthesis.     

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by enterocytes of rat small intestine.

The cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE1) is rapidly metabolized in enterocytes. The degradation of HNE and other aldehydic products of lipid peroxidation processes seems to be an antioxidative defense system. The metabolism of HNE was studied in suspensions of rat enterocytes at 37 degrees C, pH 7.4 and at initial HNE concentration of 100 microM. About 70% of the HNE were degraded within three minutes of incubation. Main products of HNE which were identified in enterocytes were the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the 1,4-dihydroxynonene. Furthermore, the formation of metabolites of the tricarboxylic acid cycle is suggested. The quantitative share of HNE binding to proteins was low with about 1% of total HNE consumption after three minutes of incubation.     

Ionic dependence of glycylsarcosine uptake by isolated chicken enterocytes

Dipeptide transport was studied in chicken enterocytes and its properties compared with those of Na+-dependent sugar transport. Results showed that 1) isolated cells were capable of accumulating glycylsarcosine (Gly-Sar) against a concentration gradient (2.5- to 3.0-fold accumulation). This uptake was maximal at pH 6.0, and it was inhibited by Na+-free medium and by ouabain; 2) uptake of Gly-Sar was not affected by methionine and was competitively inhibited by carnosine, with a Ki of 12 mM; 3) the protonophore FCCP inhibited both Gly-Sar and 3-oxy-methyl-D-glucose (3-OMG) uptake by the cells; 4) amiloride, a well-known inhibitor of the Na+/H+ exchanger system stimulated 3-OMG uptake and inhibited Gly-Sar uptake, its effects being greater at pH 7.4; 5) and monensin prevents the effects of amiloride on both sugar and dipeptide uptake. In summary, Gly-Sar uptake depends on extracellular Na+ in an indirect manner via its effect on H+ efflux, and it appears to be dependent on an inward H+ gradient.     

Isolation and characterization of chicken thymic electrolectin.

We have detected the presence of a beta-D-galactoside-binding lectin (electrolectin) in extracts of the thymus of adult chickens. This lectin was purified by affinity chromatography on a lactosyl-Sepharose column to yield 1.4 mg of pure protein from 230 g of thymus. The chicken thymic electrolectin (CTE) has an Mr of 15 300 when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of 30 000 when analysed by gel filtration. The amino acid composition of CTE is similar to that of other electrolectins purified from human and rat lung. CTE cross-reacts immunologically, but is not identical, with electrolectins from electric-eel electric organ and from chick-embryo pectoral muscle. CTE agglutinates chicken thymocytes but does not appear to promote their mitosis.     

Influence of external K+ on potassium efflux in isolated chicken enterocytes.

1. Efflux of K+ was measured in pre-loaded (86Rb+) chicken enterocytes incubated in buffers with external K+ concentration ([K+]0) between 1 and 40 mM. 2. A decrease in [K+]0 from 6 to 1 mM reduced the rate constant of K+ efflux, whereas it was stimulated by increasing [K+]0 from 6 to 40 mM. 3. The inhibitory effect of low [K+]0 on K+ efflux was: (i) higher than that expected from a change in the electrical driving force, suggesting that membrane K+ permeability has been decreased, and (ii) attenuated by A23187 and Na(+)-free buffers. 4. The effect of A23187 on K(+)-induced K+ efflux was abolished by apamin and that of Na(+)-free buffers by apamin, quinine or verapamil, which suggests that the effect of low K+ on K+ efflux seems to be due to decreased intracellular Ca2+ concentration. 5. The stimulatory effect of 40 mM K0+ on K+ exit can be accounted for by an increase in the electrical driving force. 6. The efflux of K+ at 40 mM K0 appears to occur through Ca2(+)-activated K+ channels (KCa) since it was prevented by 500 microM quinine and unaffected by bumetanide or 3,4-diaminopyridine. 7. In addition, the current results show that an increase in external K+ concentration reduced the ability of quinine to inhibit KCa channels, and even abolished that of Ba2+ and apamin.     

Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [³H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G₀ stage of the cell cycle.     

Arginine metabolism in rat enterocytes

Rat enterocytes exposed to L-arginine in the absence of any other exogenous substrate were found to actively metabolize this cationic amino acid. L-Arginine was converted to L-citrulline either directly in a NADPH-sensitive manner thought to be coupled with the generation of NO, or indirectly through the sequence of reactions catalyzed by arginase and ornithine transcarbamylase. A large fraction of L-citrulline and L-ornithine generated from exogenous L-arginine was released in the incubation medium. The production of CO2 and (poly)amines from L-arginine occurred at rates 2 to 3 orders of magnitude lower than that characterizing the net uptake of the cationic amino acid, and this despite the fact that enterocytes were equipped to allow the interconversion of L-ornithine and L-glutamate. It is concluded that the oxidative catabolism of L-arginine in enterocytes is quantitatively negligible relative to its conversion to L-citrulline and L-ornithine.     

Histidine and histamine metabolism in rat enterocytes

We have shown that the Metabolic Control Analysis (MCA) can explain the threshold effect observed in the expression of mitochondrial diseases [8]. As a matter of fact, the effect of a specific inhibitor on the flux of O2 consumption mimics a defect in a step of oxidative phosphorylation. The observed threshold is correlated to the value of the control coefficient of the inhibited step.For this reason, we have studied the repartition of the control coefficients of different steps in oxidative phosphorylation on various tissues (liver, kidney, brain, skeletal muscle and heart). We discuss the results in terms of metabolic control theory and provide a possible explanation for the heterogeneous phenotype of those pathologies. We present the double threshold hypothesis of both a threshold in the energy demand of a tissue and in the energy supply by oxidative phosphorylation. (Mol Cell Biochem 174: 143–148, 1997)     

Isolation and characteristics of carbohydrate chains of riboflavin-binding glycoprotein from the chicken egg

Isolation and characterization of oligosaccharides of riboflavin binding glycoprotein from hen white is described. Reductive cleavage of the N-glycosylamide carbohydrate-peptide bond with LiBH4/tert-BuOH followed by NaBH4-NaOH treatment gave rise to alditols, which were fractionated by means of HPLC. Twelve alditols were isolated in quantities sufficient for the monosaccharide analysis. Possibility of an ovomucoid-type oligosaccharide structure for all the alditols is discussed.     

Isolation of the lysozyme gene of chicken.

The lysozyme gene has been purified by molecular cloning from two chicken gene libraries. Several recombinant phages harbouring sequences homologous to a plasmid carrying a double stranded lysozyme cDNA have been isolated. One recombinant appears to carry an entire lysozyme gene. Electron microscopic studies show that the latter is split by at least three introns. The length of the gene is about 3.9 kb, 6 times longer than lysozyme mRNA.     

Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells

We have developed a procedure for the dispersion of mast cells from the intestinal lamina propria (LP) and epithelium of rats infected with the intestinal nematode, Nippostrongylus brasiliensis. The dispersed cells are morphologically and histochemically similar to intestinal mucosal mast cells (MMC) in situ and are distinguishable from peritoneal mast cells (PMC). MMC derived from the LP or epithelium of parasitized animals secrete histamine in response to the specific parasite antigens as well as anti-IgE. Unlike PMC, these cells are unresponsive to the basic secretagogues 48/80 and bee venom peptide 401. Similarly, bee venom peptide 401 conjugated with dansyl chloride binds to PMC and mast cells in the thymus and intestinal serosa, but not to mast cells in or derived from the intestinal LP and epithelium. Studies on PMC treated by the intestinal cell isolation procedure show that the functional characteristics of the MMC cannot be solely attributed to the isolation procedure. Thus, MMC have been isolated and shown to be morphologically, histochemically, and functionally different from PMC, as suggested by previous in vivo studies of the normal intestine.     

Subcellular localisation of di- and tripeptidases in guinea pig and rat enterocytes.

Enterocytes were isolated from rat and guinea pig jejunum and subcellular fractions were prepared by density gradient centrifugation. Gradient fractions were assayed for principal organelle marker enzymes and for di- and tripeptidases. The hydrolases showed a dual localisation with both brush border and cytosol components. In the rat, approximately equal portions of dipeptidase activities were found in the two fractions but, in the guinea pig, three times more activity were found in the two fractions but, in the guinea pig, three times more activity was found in the soluble than in the brush border fractions. Cytosol components in the rat were markedly inhibited by p-hydroxymercuribenzoate. In both species tripeptidase, leucyl-2-naphthylamidases and gamma-glutamyltransferase activities were found predominantly in the brush border fractions.     

Detection of apolipoprotein C in human and rat enterocytes

Apoproteins have important physiologic functions in lipoprotein metabolism. Several apoproteins are produced in the intestine including ApoA-I, ApoA-IV, and ApoB. Each appears to participate in intestinal lipid transport. The liver also produces several apoproteins, including ApoC-II and ApoC-III, but the data demonstrating the ability of the intestine to produce ApoC is incomplete. Our aim was to ascertain whether ApoC-II and ApoC-III were present in human and rat jejunum, and if so, whether their presence was altered by fat feeding. The technique of immunolocalization and a newly developed double antibody radioimmunoassay for rat ApoC-III3 were used. ApoC-III3 was found in the supranuclear regions of enterocytes along the entire lengths of villi in the jejuna of 12-h-fasted rats. 1 hour after the gastric ingestion of corn oil. ApoC-III3 was found primarily in between cells and in the lamina propria. Similar results were obtained in human jejunal biopsies with ApoC-II and ApoC-III. ApoC-III3 was also detected by radioimmunoassay in enterocytes isolated from jejuna of neonatal and adult rats. Thus, ApoC-II and ApoC-III are clearly present in the intestine as well as in the liver. In addition, because their localization is altered after fat feeding, they are also likely to be produced in the enterocyte.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Effects of anisosmotic buffers on K+ transport in isolated chicken enterocytes

Cells isolated by hyaluronidase incubation from chicken small intestine were used to study the effects of anisosmotic buffers on K+ transport. Hypo-osmolarity (200 mosmol.l-1) reduced both the ouabain-sensitive and the ouabain-resistant, but bumetanide-sensitive, net K+ influx and increased the K+ efflux. The hypo-osmolarity induced K+ efflux was prevented by quinine and unaffected by bumetanide. These results suggest that Ca2+-activated K+ channels may be involved in regulatory volume decrease in chicken enterocytes. Hyperosmotic conditions (400 mosmol.l-1) increased the portion of net K+ influx mediated by the Na+/K+-ATPase and that mediated by the bumetanide-sensitive K+ transport system, and decreased the K+ efflux.