Plasmid-encoded regulation of colicin E1 gene expression.

A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments. The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid. An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.     

Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.     

Histochemical demonstration of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues by means of almond glycopeptidase digestion

Almond glycopeptidase is an enzyme which cleaves specifically beta-aspartylglucosylamine linkages in glycoproteins with asialo-carbohydrate moieties. With this enzyme, it was possible to demonstrate the localization of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues. In these tissues, the oligosaccharides were shown to react positively for a series of histochemical procedures for neutral complex carbohydrates such as periodic acid-Schiff (PAS), peroxidase-labelled Ricinus communis agglutinin-I-diaminobenzidine (PO-RCA-DAB) and concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB). The asparagine-linked carbohydrates were localized in the placental villi, blood vessels and perivascular tissues and the umbilical cord blood vessels and matrix. The results of previous biochemical analyses performed upon the same tissues (Takahashi et al., 1981) have corroborated the results of the histochemical studies. The present results appear to substantiate the usefulness of almond glycopeptidase for the histochemical demonstration of the particular oligosaccharides of glycoproteins in tissues in general.     

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.     

Retention of Metabolic and Differentiation Potentials of Green Lavandula vera Callus after Freeze-Preservation

Green Lavandula vera callus that produced biotin was preservedsuccessfully in liquid nitrogen for up to 3 weeks. We foundthat the callus recovered after the freeze-preservation, retainingnot only the biosynthetic capability for biotin but also differentiationpotentials such as chloroplast development and plantlet formation.The significance of retention of the metabolic and differentiationpotentials of the callus is discussed in terms of the freeze-preservationof plant genetic resources. ^* This study is dedicated to the late Professor J. Ashida. (Received September 1, 1982; Accepted November 5, 1982)     

Isoelectric focusing with reduced cathodic drift and migration into the anode chamber

An apparatus has been developed to reduce cathodic drift and migration into the anode chamber in vertical gel rod isoelectric focusing (IEF). In contrast to commercially available apparatuses, this apparatus can easily handle many more gels at one time, and the length, diameter and shape of its gel can be arbitrarily changed. In addition, high concentrations of detergent can be used to dissolve the protein samples, and removal of the gel cylinders from the glass tubes is easy.     

Intramolecular cross-linkage of lysozyme. Imidazole catalysis of the formation of the cross-link between lysine-13 (epsilon-amino) and leucine-129 (alpha-carboxyl) by carbodiimide reaction

The salt bridge between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme was converted to an amide bond by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) reaction in the presence of imidazole (0.3-1 M) at pH 5 and room temperature, followed by dialysis at pH 10. Absence of imidazole under a similar condition did not give this intramolecularly cross-linked lysozyme derivative (CL-lysozyme) but resulted in the formation of intermolecularly cross-linked lysozyme oligomers. From the mechanistic studies on the formation of CL-lysozyme, imidazole was suggested to play the following three roles. (1) Some carboxyl groups activated by EDC in lysozyme were converted to acylimidazole groups which protected them from the reaction with amino groups in other lysozyme molecules at pH 5. These could be hydrolyzed at pH 10 to regenerate free carboxyls. (2) High concentrations of imidazole (pH 5) increased the ionic strength of the solution which weakened the salt bridge in lysozyme and facilitated the activation of the alpha-carboxyl group by EDC. (3) The alpha-carboxyl group activated by EDC was converted to an acylimidazole group which could react with the epsilon-amino group of Lys-13 in the same molecule to form an amide bond. The last step may involve some conformational change of the backbone of lysozyme and be slower than the hydrolysis reaction of the alpha-carboxyl group activated by EDC itself. However, acylimidazole groups are stable against hydrolysis at pH 5. This may afford enough time to allow the epsilon-amino group of Lys-13 to attack the acylimidazole group of Leu-129.     

Absence of direct action of insulin on metabolism of the isolated perfused rat brain

The ability of insulin to influence directly the metabolism of the mammalian brain has been evaluated with an isolated, perfused rat brain preparation. Insulin was added to the perfusion fluid or was injected into the rat from which the isolated brain preparation was subsequently made. The spontaneous electrical activity of the brain, the rate of cerebral glucose consumption and the rate of efflux of K⁺ from the brain were not affected by insulin. We conclude that insulin either does not act directly on the brain or that its action is very small and/or very slow in comparison with its action on other tissues. We suggest that the effects on brain metabolism reported to occur after administering insulin and glucose to the intact animal may be secondary to the large stimulation of the metabolism of the liver and/or other organs.