Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Interaction of glycylglycine and Na+ at the mucosal border of Guinea-pig small intestine: A non-mutual stimulation of transport

Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na⁺ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na⁺, while Na⁺ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PD_t), short-circuit current (I_sc) and Na⁺ influx. The change in PD_t (ΔPD_t) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between I_sc and Na⁺ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na⁺, and that translocation of Na⁺ occurs when the two Na⁺ sites on the carrier loaded with Gly-Gly are occupied by Na⁺. However, our results indicate that the resultant Na⁺ flow is not capable of stimulating Gly-Gly translocation.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

The interaction of phagocytes and the large-sized parasite Cryptococcus neoformans: Cytochemical and ultrastructural study

Summary The yeast Cryptococcus neoformans may develop under certain conditions a large polysaccharide capsule 50–100 M in diameter and therefore cannot be phagocytosed by either polymorphonuclear cells (PMN's) or mononuclear phagocytes (MN's). The cellular defense mechanism — in various animals — against the yeast is composed by formation of ringlike structure of PMN's or MN's cells which surround the C. neoformans. Ring structures develop either in vivo or in vitro in tissue culture; destruction of the yeast occurs within 36–72 hours.Several hydrolases, such as acid phosphatase, -glucuronidase and non-specific esterase were found to be released from the phagocytic cells into the enclosed yeast. Considerable reduction of NBT used as a marker for oxidative activity was observed in MN rings at contact regions of the MN cells and the yeast. Electron microscopic studies indicate that the phagocytic cells in the ring structure have many pseudopodes penetrating into the polysaccharide capsule of the yeast. Disintegration of the capsule was observed as well as phagocytosis of its material. A possible analogy between normal phagocytosis of small-sized bodies and the ring structure obtained when large bodies are involved is discussed.     

掷孢菌科的研究——Ⅲ.不同掷孢酵母属产掷孢子的能力及担子菌酵母形成特大厚垣孢子的规律

本文以分离自中国的大量掷孢酵母为材料,研究了不同属的掷孢酵母生掷孢子的能力,以及此能力的持久性,发现属间差异很大。此外还报道了142株不同酵母的产厚垣孢子的情况,发现与担子菌有关的4属酵母在接近致死温度时,都会出现特大厚垣孢子如红掷孢酵母(Sporobolomyces),布勒掷孢酵母(Bullera),红酵母(Rhodotorula)与隐球酵母(cryptococcus),而属于子囊菌的酵母属(Saccharomyces)则无此现象,从而对某些接近担子菌的酵母又增加了新的认识。     

Systematics of theLactobacillus population on rat intestinal mucosa with special reference toLactobacillus reuteri

The systematics of theLactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-twoLactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains ofLactobacillus andLeuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% S_J-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains ofL. jensenii, L. gasseri, L. crispatus, and to some extent toL. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified asL. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of theL. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.     

Processing and transfer of epidermal growth factor in developing rat jejunum and ileum

Using everted sac technique we demonstrated the transfer of ¹²⁵I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline. RP-HPLC analysis detected carboxy-terminal processing of ¹²⁵I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced ¹²⁵I-des(53)mEGF and ¹²⁵I-des(49–53)mEGF, whereas ¹²⁵I-des(48–53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of ¹²⁵I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of ¹²⁵I-des(53)mEGF was higher than that of ¹²⁵I-mEGF, but those of ¹²⁵-des(49–53)mEGF and ¹²⁵I-des(48–53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Immunochemical and immunohistological expression of Lewis histo-blood group antigens in small intestine including individuals of the Le(a+b+) and Le(a−b−) nonsecretor phenotypes

Histological samples and total non-acid glycosphingolipids were prepared from small intestine of human cadavers with the Le(a+b+) and Le(a–b–) nonsecretor phenotypes and contrasted with the more common Lewis phenotypes. Glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with monoclonal antibodies reactive to Lewis epitopes. Paraffin-embedded small intestine sections were also fluorescently immunostained with anti-Lewis antibodies. Unlike the common Lewis positive phenotypes, we were immunochemically able to demonstrate the copresence of large amounts of Le^a and Le^b glycolipids in the Le(a+b+) sample. In addition we demonstrated increased formation of extended Lewis structures in this phenotype. By immunohistochemistry Le^a, Le^b and type 1 precursor chain epitopes could be demonstrated in the brush border. These results show that the expression of the Le(a+b+) phenotype at the erythrocyte phenotyping level parallels the small intestinal expression of this phenotype, and the patterns of Lewis antigen expressions are unique to this phenotype. By immunohistochemistry and immunochemistry we also demonstrated the presence of trace amounts of Lewis active glycoconjugates in the small intestine of the Le(a–b–) nonsecretor and Le(a+b–) samples. In the Le(a–b–) nonsecretor Le^a and Le^b activity was absent and type 1 precursor was present in brush border, while Le^b activity was immunohistologically demonstrated in the Golgi apparatus of the deep glands. Trace amounts of both Le^a and Le^b glycolipids were identified in this sample. In parallel trace Le^b activity could also be detected in the glycolipids of the Le(a+b–) sample and could be immunohistologically demonstrated to be fully expressed in occasional cells in the deep glands of the small intestine, a pattern quite dissimilar to that of the Le(a–b–) nonsecretor. The results in this paper show that the expression of Lewis glycoconjugates in the small intestine parallel the expression of Lewis erythrocyte phenotypes. However, inappropriate Lewis activity is also seen in individuals of other phenotypes and the mechanisms by which these Lewis antigens are made appears to be different for different phenotypes.Abbreviations FITC fluorescein isothiocyanate - HPLC high-performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - RBC red blood cell - TLC thin-layer chromatography - TRITC tetramethyl rhodamine isothiocyanate