A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Binding of staphylococcal cell surface polysaccharide to human fibrinogen

The interaction between the binding site of a polysaccharide (called compact colony forming active substance (CCFAS)), obtained from the cell surface of a strain of Staphylococcus, and human fibrinogen (HF) was investigated. The CCFAS was found to bind specifically to both the B beta and gamma chains of HF at pH 7.0 and 8.0, and the A alpha chain at pH 5.0. The binding of CCFAS with fibrinogen fragments obtained by digestion with plasmin were also investigated. Fragments with Mr of 55,000, 24,000, and 19,000 were the major bands precipitated by CCFAS at pH 7.0 and 8.0. Fragments with Mr of 85,000 and 75,000 bound to CCFAS at pH 5.0. Binding of CCFAS (7 micrograms) with fibrinogen could be inhibited by 1.2 micrograms of B beta chain and 1.5 micrograms gamma chain at alkaline pH or 6.2 micrograms of the A alpha chain at pH 5.0. CCFAS was, therefore, assumed to be specifically bonded with HF molecules, in the alkaline range at least, resulting in compact colony forming activity in serum soft agar and paracoagulation.     

Blood vascular architecture of the rat lingual papillae with special reference to their relations to the connective tissue papillae and surface structures: a light and scanning electron microscope study

Subepithelial blood vessels of the rat lingual papillae and their spatial relations to the connective tissue papillae and surface structures were demonstrated by light and scanning electron microscopy. In the rat, four types of papillae were distinguished on the dorsal surface of the tongue, i.e. the filiform, fungiform, foliate and circumvallate papillae. Vascular beds of various appearance were found in all four types of lingual papillae: a simple or twisted capillary loop in the filiform papilla; a basket- or petal-like network in the fungiform papilla; a ring-like network in the foliate papilla, and a conglomerated network surrounded by double heart-shaped capillary networks in the circumvallate papilla. These characteristic vascular beds corresponded to the shape of the connective tissue papillae and surface structures. The vascular bed beneath the gustatory epithelium in the fungiform, foliate and circumvallate papilla consisted of fine capillary networks next to the taste buds.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Effect of short — chain fatty acids on electrical activity of the small intestinal mucosa of rat

Intravenous administration of short-chain fatty acid (SCFA), such as propionate, butyrate, valerate and caproate, caused a transient increase in transmural potential difference (p.d.) across the small intestine of rat $\text{in vivo}$. There was a sigmoid relationship between the change in the p.d. and the logarithm of the dose of SCFA. The median effective dose of propionate, n-butyrate, n-valerate and n-caproate, which was calculated from the each dose-response curve obtained from the terminal ileum, 1.31, 1.43, 0.83 and 0.81 μmole, respectively. Repeated administrations of the same dose of propionate evoked progressively smaller response. The dose-response curve of propionate was shifted to the left by neostigmine and to the right by atropine, suggesting that the action of SCFA may be mediated by acetylcholine, which was released from a nerve ending.     

Catenation and supercoiling in the products of bacteriophage λ integrative recombination in vitro

Catenanes (interlocked circular DNA molecules) are the exclusive products of the bacteriophage λ integrative recombination reaction in vitro when the substrate is a supercoiled DNA molecule containing both the attP and attB sites. It is proposed that the catenation results from the superhelical form of the substrate DNA. We also show that both circular DNA products of a single recombination event can be recovered as superhelical molecules with a superhelical density approximately that of the substrate DNA. The recombination reaction must therefore occur as a coupled process which does not permit free rotation around single-strand breaks at any stage.