Relative effects of cooling and warming rates on mammalian cells during the freeze-thaw cycle

The relative roles of cooling and warming rates on cell survival during a freeze-thaw cycle were investigated. Basically the faster the warming rate, the better the cells survive. One of the factors influencing this is the extended phase transition period at the slower thawing rates. The warming rate had a significant effect on cell damage and recovery, but this was not as great as comparative changes in the cooling rate were. This investigation also showed that under certain freeze-thaw conditions there was a lack of correlation between the two methods used for quantifying cell recovery (RI) and cell damage (PCR) as measured by radiochromate release. The analysis of the relationship between RI and PCR showed that PCR could be used to measure both lethal and nonlethal damage and enabled a clearer interpretation of cellular damage during cooling and thawing to be made.     

Rat hepatic uroporphyrinogen III cosynthase: Purification,properties, and inhibition by metal ions

Rat hepatic uroporphyrinogen III cosynthase has been isolated and purified 50-fold with a 36% yield by ammonium sulfate fractionation and sequential chromatography on DEAE-Sephacel and Sephadex G-100SF. Inhibition of uroporphyrinogen III formation with increasing porphobilinogen concentration was observed. Cosynthase was shown to be thermolabile, and a time-dependent loss of enzyme activity during reaction with uroporphyrinogen I synthase and porphobilinogen was observed. The pH optimum for the complete system (synthase and cosynthase) was pH 7.8 in 50 mm Tris-HCl or 50 mm sodium phosphate buffer. Various metals (KCl, NaCl, MgCl₂, CaCl₂) increased formation of Uroporphyrinogen III. Heavy metals including ZnCl₂, CdCl₂, and CuCl₂ were shown to selectively inhibit cosynthase activity, whereas other metals (HgCl₂, PbCl₂) were less selective and inhibited both synthase and cosynthase at similar concentrations.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

An in vitro model for induction of immunologic unresponsiveness to turkey gamma-globulin in primed mouse spleen cells, II. Antigen-specific suppressor cells.

Unresponsiveness induced to turkey γ-globulin (TGG) in cultures of TGG-primed spleen cells by incubation with high concentrations of soluble TGG (sTGG) was shown to involve a state of active suppression. Upon transfer to secondary cultures of primed spleen cells stimulated with an optimal dose of TGG-conjugated erythrocytes, such tolerant spleen cells were able to actively inhibit a secondary plaque-forming cell response to TGG in these cultures. Almost complete inhibition was observed with a tolerant cell to primed cell ratio of as low as 0.1. The suppression was antigen specific in that tolerant spleen cells which were suppressive for the secondary TGG response were unable to inhibit a primary response to sheep erythrocytes. T cells were shown to be required for the suppressor effect, in that (i) suppressor activity could be removed by complement-mediated lysis with an anti-Thy 1.2 antiserum and (ii) suppressor activity was retained in the effluent fraction after passage of suppressor spleen cells over a nylon wool column. Induction of the T-cell suppressor activity was found to be associated with a loss of T-cell helper activity within the TGG-pulsed cell population. The presence of adherent cells was not required for induction of suppressor activity. Furthermore, the suppressor effect was found to be resistant to 1000 R of γ irradiation.     

An improved treatment of data for estimation of molecular weights by sephadex gel filtration

A method for analysis of elution data of proteins, obtained from Sephadex gel filtration experiments, is described. The relevant elution data from seven different proteins, with known molecular weights and Stoke's radii, were fitted into various equations relating elution parameters and molecular size parameters. It was observed that polynomial relationships represented elution data for proteins with a much greater degree of precision than linear equations. The validity of this procedure was also checked by analysing gel filtration data available in the literature and it was concluded that a better fit was obtained using polynomial relationships, provided a sufficiently large number of experimental points were available for numerical analysis. Using this method, values of 320,000 ± 7000 for the molecular weight, and (60 ± 0.4) × 10^?8 cm for the Stoke's radius of Neurospora NAD-specific glutamate dehydrogenase were calculated.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Regulation of energy metabolism in Saccharomyces cerevisiae. Relationships between catabolite repression, trehalose synthesis, and mitochondrial development.

Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cerevisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetase-trehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (ρ^?) mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.     

Biochemical,immunological, and structural studies on a sphingolipid activator protein (SAP-1)

Sphingolipid activator protein-1 (SAP-1) is a glycoprotein found in human tissue extracts that stimulates the enzymatic hydrolysis of at least two glycosphingolipids, including GM1 ganglioside and sulfatide. The ability of purified SAP-1 to stimulate GM1 ganglioside hydrolysis by extracts of cultured fibroblasts from patients with β-galactosidase deficiency was examined, and all patients had a pronounced deficiency (under 10% of control). Using monospecific antibodies against SAP-1, the concentration was determined in cultured fibroblasts by rocket immunoelectrophoresis. Extracts from 15 control cell lines were found to have 0.72 ± 0.24 μg cross-reactive material/mg protein, while cell extracts from 8 patients with GM1 gangliosidosis involving mental retardation were found to have 1.08 ± 0.17, which is significantly elevated. When the fibroblast extracts were subjected to sodium dodecyl sulfate-polyacramide gel electrophoresis followed by electroblotting, multiple bands were observed. Controls were found to have two major bands with estimated molecular weights of 9000 and 9500, and a minor band at 7800. Extracts from patients with GM1 gangliosidosis were found to have multiple bands ranging upward to 13,000. Extracts from patients with the most severe clinical types of GM1 gangliosidosis had almost exclusively high-molecular-weight forms (molecular weights above 10,000). Treatment of SAP-1 from control liver with endoglycosidase D caused a decrease in the M_r 9500 band and increased in the M_r 7800 band. When SAP-1 from GM1 gangliosidosis liver was treated sequentially with neuraminidase, β-galactosidase, and endoglycosidase D, almost all of it was converted to the forms found in control human liver.     

An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher)

The twitcher mouse is genetically determined mutant characterized by a deficiency of galactocerebroside beta-galactosidase. In this study, a significant accumulation of galactocerebroside was demonstrated in twitcher mouse kidney. The data suggest that mouse Krabbe's disease is not only involved in CNS, but also in visceral organs.     

Role of silicon in diatom metabolism : X. Polypeptide labelling patterns during the cell cycle, silicate starvation and recovery in Cylindrotheca fusiformis

The soluble polypeptides from Cylindrotheca fusiformis were labelled with [³⁵S]O₄²⁻ and resolved by two-dimensional gel electrophoresis. More than 600 polypeptides were detected upon a 26-day exposure to X-ray film. Analysis of the labelling pattern during the cell cycle show that labelling of at least 208 polypeptides changes; the majority, however, remain unchanged. Most of the changes occur in the beginning of the cell cycle and typically involve increases; those occurring in the second half of the cycle typically involve decreases. Light or its absence affects apparent protein turnover and the labelling rates of several polypeptides. Polypeptide labelling during the cell cycle was used as a reference to analyse the effect of silicate deprivation on diatom metabolism. In the absence of silicate, protein turnover increases: however, the addition of silicate counteracts but does not fully reverse this change. Silicate starvation affects the program of synthesis for several polypeptides, but in general the program of polypeptide labelling continues up to the S phase of the cell cycle. Addition of silicate to silicate-starved cells causes the appearance of four hitherto undetected polypeptides.     

The generation of memory B-cell subpopulations capable of proliferation and expansion of the pool: effect of time and antigen

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.     

Simultaneous single isotope radioenzymatic assay of plasma norepinephrine,epinephrine and dopamine

Modification of the original single isotope radioenzymatic assay of Passon and Peuler (1) permits the direct and simultaneous analysis of norepinephrine, epinephrine and dopamine in plasma samples of 50 μl or less. Plasma or cerebrospinal fluid without prior extraction of catecholamines or deproteinization is added directly into a mixture of 100 μl. This catechol-O-methyl-transferase-catalyzed assay is sensitive to 1 pg (20 pg/ml of plasma) for norepinephrine and epinephrine and 6 pg (120 pg/ml) for dopamine. A rapid thin layer chromatographic separation of the three ³H-methylcatecholamines contributes to the excellent specificity of the differential assay of the three catecholamines. The differential analysis of 15–20 plasma samples can be completed easily within one day. A total assay which omits the chromatographic step and, thus, measures norepinephrine plus epinephrine at the same sensitivity can be completed in 20 samples in one-half a working day.     

Program of early development in the mammal: changes in the patterns and absolute rates of tubulin and total protein synthesis during oocyte growth in the mouse

Oocytes at several stages of growth have been isolated by enzymatic digestion and/or physical disruption of ovaries excised from juvenile and adult mice. The absolute rates of total protein synthesis and tubulin synthesis in these isolated oocytes were determined by measuring sizes of the endogenous methionine pool and apparent rates of incorporation of [³⁵S]methionine into total protein and tubulin using methods described previously (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341). The size of the endogenous methionine pool increases approximately 350-fold during oocyte growth, from 0.16 fmole in nongrowing oocytes (12 μm) to 56 fmole in fully grown oocytes (85 μm). Since the volume of mouse oocytes also increases about 350-fold during growth, the concentration of intracellular free methionine remains constant at approximately 170 μM. The absolute rate of protein synthesis increases from 1.1 to 41.8 pg/hr/oocyte for nongrowing and fully grown mouse oocytes, respectively. Since this represents about a 38-fold increase in the absolute rate of protein synthesis, the rate of synthesis per picoliter of cytoplasm actually decreases nearly 10-fold during oocyte growth. These measurements indicate that the growing mouse oocyte itself is capable of synthesizing only about 50% of the protein found in fully grown oocytes. Tubulin is one of the major proteins synthesized by growing mouse oocytes since the absolute rate of tubulin synthesis is, on the average, 1.8% of total protein synthesis. The absolute rate of tubulin synthesis increases from 0.4 to 0.6 pg/hr/oocyte as the oocyte grows from 40 to 85 μm in diameter. However, overall, the percentage of total protein synthesis devoted to the synthesis of tubulin actually declines somewhat during this phase of growth, from 2 to 1.5%. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of absolute rate of synthesis of the β subunit to that of the α subunit varies from 1.3 to 2.0 throughout oocyte growth. High-resolution two-dimensional gel electrophoretic analyses of [³⁵S]methionine-labeled proteins reveal that many changes take place in the pattern of protein synthesis during oocyte growth.     

Bull sperm adenylate cyclase: localization and partial characterization.

A purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from bovine thyroid tissue has been purified 670-fold utilizing the techniques of ammonium sulfate precipitation, ion-exchange and molecular-exclusion chromatography, and polyacrylamide-gel electrophoresis. The protein has an apparent molecular weight of 90,000, a single isoelectric point at 5.6, and a Michaelis constant of 0.028 mm for inosine. Double-reciprocal plots of the reaction rate for the phosphorylase-catalyzed reaction versus phosphate or arsenate concentration display a downward trend at high substrate concentrations. Two apparent Michaelis constants of 0.38 and 1.49 mm were determined for phosphate.     

Superoxide dismutase from Bacillus stearothermophilus: Crystallization and preliminary X-ray diffraction studies

Superoxide dismutase from Bacillus stearothermophilus is a dimeric manganese-containing enzyme with a molecular weight of 40,000. It has been crystallised in the monoclinic space group P2₁ with unit cell dimensions of $\text{a = 50}\text{A}\text{?}\text{, b = 70}\text{A}\text{?}\text{, c = 69}\text{A}\text{?}$ and β = 111 ° 10′. The asymmetric unit appears to be the dimer.     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.     

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The V_max for the demethylation reaction exhibited a pH optimum at approximately 4.5. The K_m for N,N-dimethylaniline increased with decreasing pH, while the K_m for ethyl hydroperoxide varied in a manner paralleling V_max. Comparison of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the V_max versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.     

The rabbit pulmonary monooxygenase system: characteristics and activities of two forms of pulmonary cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450 have been characterized spectrally and their activities in reconstituted monooxygenase systems investigated. The presence of both microsomal phospholipids and sodium cholate was required to obtain optimum activity. Only one of the cytochromes (I) was active in the N-demethylation of benzphetamine and the O-deethylation of 7-ethoxycoumarin. However, cytochrome II was 20% more active than cytochrome I in the metabolism of benzo[a]pyrene. The profile of the metabolites formed from benzo[a]pyrene indicated that metabolism at the 9 and 10 positions was insignificant in the case of cytochrome I but represented about 40% of the metabolites produced by cytochrome II. The two forms of the cytochrome are present in pulmonary microsomes in approximately equal amounts.