Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.     

L-649,923, sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propylthio)- gamma-hydroxy-beta-methylbenzenebutanoate, a selective, orally active leukotriene receptor antagonist

L-649,923, Sodium (beta S*, gamma R*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)propylthio)- gamma- hydroxy-beta-methylbenzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (Ki value of 400 nM) and to a lesser extent [3H]leukotriene C4 (Ki value of 8.6 microM) binding in guinea-pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotriene C4, D4, E4, and F4 but not those induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (stable endoperoxide analogue). Schild plot analysis indicated a competitive inhibition of contractions of guinea-pig ileum induced by leukotriene D4 (pA2 8.1) and contractions of guinea-pig trachea induced by leukotrienes E4 and F4 (pA2 7.1 and 6.9, respectively). In contrast, contractions of guinea-pig trachea induced by leukotrienes C4 (pA2 7.2; slope 0.6) and D4 (pA2 7.2; slope 0.7) were inhibited in a noncompetitive fashion. In vivo, intravenously administered L-649,923 selectively blocked bronchoconstriction induced in anesthetized guinea pigs by leukotriene C4 and D4 (ED50 values i.v. 0.38 and 0.26 mg/kg, respectively) but not that induced by histamine, arachidonic acid, serotonin, U-44069, or acetylcholine. Following intraduodenal administration, L-649,923, blocked leukotriene D4 induced bronchoconstriction (5 and 10 mg/kg). The present findings indicate that selective antagonists, such as L-649,923, may be useful for defining the role of leukotrienes in diseases such as bronchial asthma.     

Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver

In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.     

Treatment of dialysis membranes for simultaneous dialysis and concentration

Dialysis membranes used for simultaneous dialysis-concentration required pretreatment to remove uv-absorbing compounds leached from the membranes and to reduce the absorption of protein to the membranes. This was accomplished with sodium carbonate and ethanol or with "sulfur-removal solutions." Protein determinations were made with a micro-Bradford protein reaction and with uv absorbance at 280 nm. Soluble membrane components contributed to aberrant uv spectra and altered the ratio of 280/260-nm absorbance. Simultaneous dialysis and concentration in the micro protein dialyzer-concentrator apparatus, combining aspects of thin-layer dialysis and ultrafiltration, resulted in rapid removal of salts from the protein solutions. Prior treatment of membranes reduced uncertainties in retentate recoveries, eliminated uv-absorbing components of membranes, and improved recoveries of protein.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization

Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.     

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

Demonstration of a proteolytic activity on intracellular origin in Aeromonas hydrophila LP 50

Aeromonas hydrophila LP 50, isolated from packaged pasteurized milk, was grown in glucose-polypeptone medium at 30 degrees C. The proteolytic activity of A. hydrophila LP 50, optimum at the stationary phase of growth, is attributed to extracellular or membrane protease; no intracellular proteolytic activity was shown.