Parametric sensitivity of stoichiometric flux balance models applied to wild-type Escherichia coli metabolism

The esterification of lauric acid with geraniol catalyzed by the commercially immobilized lipase preparation from Mucor miehei, Lipozyme(R), was studied in well-stirred flasks. The enzyme support was characterized in terms of its internal and external surface area, protein location, and protein content. It was found that the enzyme was mainly located on the external surface of the support, therefore, internal diffusional limitations were not important. It was also shown that the protein content of the support depends on the size of the particle, with smaller particles containing higher amounts of protein per unit weight. Under the conditions studied, the reaction was not under external mass transfer limitations, and the initial reaction rate depended on the size of the support particles. This was mainly due to the different protein contents on the support as a function of particle size and not to internal or external mass transfer limitations. Also, it was found that the inhibition exerted by water was predominantly a physical effect due to its accumulation around the enzyme. It was also found that the reaction was substrate inhibited by lauric acid, but not by geraniol. (c) 1995 John Wiley & Sons, Inc.     

GTPγS restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells

Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finchet al., J Biol Chem 268, 5823–5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTPS, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTPS still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.     

Fermenter production of an artificial fab fragment,rationally designed for the antigen cystatin,and its optimized crystallization through constant domain shuffling

The synthetic antibody model “M41” was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C_K and C_H 1_γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2₁2₁2₁ with unit cell dimensions a = 104.7 Å, b = 113.9 Å, c = 98.8 Å and diffracted X-rays to a nominal resolution of 2.5 Å. © 1995 Wiley-Liss, Inc.     

Cadaverin ist ein Zwischenprodukt der Biosynthese von Arthrobactin und Ferrioxamin E

Zusammenfassung Cadaverin wird mit einer höheren Ausbeute als Lysin in Arthrobactin von Arthrobacter pascens und Ferrioxamin E von Streptomyces glaucescens eingebaut. Von einem racemischen Lysingemisch kann nur das L-Isomere eingebaut werden. Die Aktivität der L-Lysindecarboxylase wurde in vivo und in vitro gemessen. Das Enzym ist durch Lysin nicht induzierbar und wird durch 5·10^-6 M Fe³⁺ vollständig reprimiert. Bei Klebsiella pneumoniae, dem Produzenten von Aerobactin, konnte nur eine geringfügige Lysindecarboxylase-Aktivität nachgewiesen werden.

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Cadaverin is an intermediate in the biosynthesis of arthrobactin and ferrioxamin E

Cadaverin was more readily incorporated than lysine into arthrobactin from Arthrobacter pascens and into ferrioxamin E from Streptomyces glaucescens. From a racemic mixture only the L-isomer of lysine is incorporated. The L-lysine decarboxylase activity was measured in vivo and in vitro. The enzyme from Arthrobacter pascens is not inducable by lysine and completely repressed by 5·10^-6 M Fe³⁺. In Klebsiella pneumoniae, the producer of aerobactin, only a very low activity of L-lysine decarboxylase was detected.

     

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing.The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane.The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow freeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures.The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.     

Material transport within specialised ciliary shafts on Rhabdopleura zooids

Summary The surface of the Rhabdopleura zooid is ciliated. The cilia of the cephalic shield and tentacles have paddle-like swellings of the shaft. These swellings are usually about 0.6–1 m in diameter and most frequently found in the distal 1–2 m of the ciliary shaft. Others are found in other positions along the length of the cilium and it is suggested that at least some of these swellings represent material transport within the cilium.Paddle shaped cilia are probably more efficient than normal cilia in moving water and food particles. If these cilia are involved in the building of the tubular coenecium then their distribution suggests that the tentacles as well as the cephalic shield are actively involved in tube building.I should like to thank the director and staff of the Marine Biological Laboratory, Plymouth, for collecting the material and the generous loan of facilities during the preparation of the material. Mr. R. Moss provided skillful technical and photographic assistance     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.