Low-serum medium development for human diploid fibroblast microcarrier cultures

A new medium supplement mixture, PPRF92, has been developed to enable the serial subculture of human diploid fibroblasts (MRC-5 cells) on microcarriers. Furthermore, the PPRF92 supplements enable cell growth at serum levels as low as 1%. Through an optimization programme, the PPRF92 supplements have evolved into a simple mixture with the concentrations of key components at a level that makes the overall cost very competitive with medium containing 10% foetal bovine serum (FBS). Furthermore, the PPRF92 supplement mixture is most efficacious when FBS is replaced with the cheaper, and more widely available, adult bovine serum (ABS). Although medium exchange with serum is necessary in order to achieve confluence on microcarriers, the PPRF92 mixture is only necessary at the initiation of each passage. Using the medium replinishment protocol that has been developed in our laboratory, MRC-5 cells were successfully serially passaged through 13 bead-to-bead transfers on microcarriers in DMEM/F12 medium enriched with the PPRF92 supplement mixture reported here, and 1% ABS.Correspondence to: L. A. Behie     

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.     

Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.     

Morganella morganii urease: purification, characterization, and isolation of gene sequences

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.     

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 mol 100 g^-1 Chl d^-1 (2 mol g^-1 FW d^-1), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K⁺ on a tissue water basis remained constant at 200 mM while Cl^- and Na⁺ levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg^-1. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [¹⁴C]serine, ethanolamine and choline (all 1 mol, 13.3 MBq in 10 l) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [¹⁴C]choline and [¹⁴C]ethanolamine was incorporation into glycinebetaine while less ¹⁴C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [¹⁴C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [¹⁴C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342–348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.Abbreviations BHT butylated hydroxytoluerte (2,6-di-tert-butyl-4-methylphenol) - C₁ one-carbon fragment - 1,2DG diglyceride moiety - DW day weight - MCW methanol-chloroform-water (12:5:1, by vol.) - PA phosphatidic acid - PC phosphatidyl choline - PMME phosphatidyl monomethylethanolamine - PDME phosphatidyl dimethylethanolamine - PE phosphatidyl ethanolamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis(5-phenyloxazoyl) benzene     

Management of massive pulmonary embolism after jejuno-ileal bypass for morbid obesity

Four patients developed massive pulmonary embolism after jejuno-ileal bypass for morbid obesity. All patients were in Greenfield's Class IV and were in shock. Severe hypoxia was evidenced in their blood gases. The patients were managed with digitalis, diuretics, Solu-Medrol (methylprednisolone sodium succinate), oxygen, and heparin therapy. Each patient underwent partial vena cava interruption with Mobin Uddin's umbrella, and all four survived without residual complications.     

Identification of the Low Molecular Weight Copper Protein from Copper-intoxicated Mung Bean Plants

Mung bean plants (Wilczek) accumulate increasingly greater amounts of buffer-extractable copper in both their shoots and roots when grown in liquid medium containing greater than 2 micrograms per milliliter copper (31.4 micromolar) as cupric sulfate. This increase in soluble copper is accompanied by an increase in the relative amount of low molecular weight (7,000 to 20,000) macromolecular-bound copper and a decrease in the relative amount of high molecular weight (greater than 20,000) copper. The major low molecular weight copper protein has been isolated from copper-intoxicated mung bean plants by a combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. It was identified as mung bean plastocyanin on the basis of its molecular weight, optical behavior, and amino acid composition. No evidence was found for a low molecular weight copper-binding protein corresponding to mammalian thionein or chelatin.     

Cell junctions and intercellular communication

Summary We have compared intercellular communication in normal and regenerating rat liver. Gap junctions are greatly reduced in size and numbers 29 to 35 hr after hepatectomy, but we still find some 90% of hepatocytes coupled by electrophysiological criteria. The spread of dyes such as carboxyfluorescein however is very limited in the regenerating organs as compared to the situation in the controls. We show how the apparent discrepancies between morphological and physiological data can be reconciled. We also present a summary of preliminary findings on the biosynthesis of gap junction protein and some of the conclusions one can draw from the sequence of 58 amino acids at the amino terminal of the protein. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. The original research described was supported by Grants GM 06965 and RR 07003 from the National Institute of Health, and funds from the North-west Area Foundation. David Meyer and Barbara Yancey were the recipients of NIH postdoctoral fellowships (NS 06240 and AM05700). This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center.     

Cytosolic phospholipase A2 from U937 cells: size of the functional enzyme by radiation inactivation.

We have studied the cytosolic phospholipase A2 (cPLA2) of human U937 cells by radiation inactivation in order to characterize the functional form of the native enzyme by a method that was independent of the discrepancies observed by SDS-PAGE and cDNA cloning. The Radiation Inactivation Size of cPLA2 was reproducible and gave a value of 76,800-80,100 daltons. We eluted the active enzyme from polyacrylamide-gradient gel electrophoresis at a molecular weight of 77,000, confirming the irradiation result. We conclude that cPLA2 is active as the monomeric enzyme and is composed of a single major functional domain that is sensitive to irradiation.