Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Changes in mucus glycoprotein synthesized in rat gastric mucosa exposed to ethanol

The resistance to proteolysis by pepsin of gastric mucus glycoprotein synthesized by tissue culture in the presence and absence of 0.1 M ethanol was investigated. The glycoprotein product of ethanol-supplemented culture was found to contain 68% less associated lipids and 81% less covalently bound fatty acids, but exhibited unaltered content of carbohydrate and protein. The lipid and fatty acyl deficient glycoprotein was 5-times more rapidly and 2-3-times more extensively degraded by pepsin than the glycoprotein synthesized in the absence of ethanol. Following delipidation with organic solvents and deacylation with hydroxylamine both glycoproteins were digested at the same rate and degraded to the same extent. The lower content of fatty acyl residues markedly affected the overall pattern of the proteolytic fragments identified by SDS gel electrophoresis. The peptides corresponding to the acylated fragments of control were degraded and an increase in the amount of smaller peptides was observed. The in vitro assays of the fatty acyltransferase activity towards the substrates obtained from control and alcohol-containing cultures revealed that the enzyme activity was similar and increased proportionally with increased concentration of both glycoprotein substrates and enzyme. However, addition of 0.1 M ethanol to the assay tubes containing complete incubation mixture decreased the acylation of either glycoprotein by 40%. Based on the results presented here, and on previous studies of mucus glycoprotein synthesis in the presence of ethanol, we conclude that ethanol interferes with the process of acylation of mucus glycoprotein with fatty acids.     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

The Roles of Sodium and Potassium Ions in the Generation of the Electro-Olfactogram

In order to clarify whether or not the electronegative olfactory mucosal potentials (EOG) are generator potentials, the effects of changed ionic enviroment were studied. The EOG decreased in amplitude and in some cases nearly or completely disappeared, when Na⁺ in the bathing Ringer solution was replaced by sucrose, Li⁺, choline⁺, tetraethylammonium⁺ (TEA), or hydrazine. In the K⁺-free Ringer solution, the negative EOG's initially increased and then decreased in amplitude. In Ringer's solution with increased K⁺, the negative EOG's increased in amplitude. When K⁺ was increased in exchange for Na⁺ in Ringer's solution, the negative EOG's decreased, disappeared, and then reversed their polarity (Fig. 6). Next, when the K⁺ was replaced by equimolar sucrose, Li⁺, choline⁺, TEA⁺, hydrazine, or Na⁺, the reversed potentials recovered completely only in Na⁺-Ringer's solution, but never in the other solutions. Thus, the essential role of Na⁺ and K⁺ in the negative EOG's was demonstrated. Ba⁺⁺ was found to depress selectively the electropositive EOG, but it hardly decreased and never increased the negative EOG. Hence, it is concluded that Ba⁺⁺ interferes only with Cl^- influx, and that the negative EOG's are elicited by an increase in permeability of the olfactory receptive membrane to Na⁺ and K⁺, but not to Cl^-. From the ionic mechanism it is inferred that the negative EOG's are in most cases composites of generator and positive potentials.     

Electrical Response and Growth of Olfactory Cilia of the Olfactory Epithelium of the Newt in Water and on Land

A correlation between the length of the olfactory cilia and the electrical activity of the olfactory epithelium was studied in newts living in water and on land. The olfactory cilia grew when newts were transferred onto land. The cilia in the olfactory bud became longest in 108 hours after the transfer and then became shorter, while those in the interstitium only gradually elongated. Slow potentials were evoked in the epithelium by the application of odorous fluids but not by odorous vapors for 20 hours after the transfer. Thereafter, the slow potential began to appear in response to odorous vapors and reached maximal magnitude between 60 and 70 hours after the transfer, while it was not evoked by odorous fluids in this period. In the later stage, the slow potential to odorous vapors decreased in magnitude and disappeared 120 hours after the transfer, while it began to reappear in response to odorous fluids. When these changes in the slow potential were compared with those in the cilium, a discrepancy was found between the period of maximal potential magnitude and that of maximal cilium length.     

Direct activation of phospholipase A2 by GTP-binding protein in human peripheral polymorphonuclear leukocytes.

In human peripheral polymorphonuclear leukocyte (PMN), 10% of PLA2 activity was found in the particulate fraction. In the particulate fraction, the activity of phospholipase A2 was enhanced 270% by 100 microM guanosine 5'-[gamma-thio]triphosphate, a hydrolysis-resistant analog of GTP. In the soluble fraction, such enhancement was not observed. Guanosine 5'-[beta-thio]diphosphate (2 mM), which irreversibly inactivates GTP-binding protein, blocked the enhancement in the particulate fraction. Membrane-binding phospholipase A2 activity of PMN would thus appear to be regulated directly by GTP-binding protein.     

Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.