Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Cellular subpopulations in the expression of IgG1.

Consistent with the surface display of IgG₁, antigen-primed B cells are sensitive to functional elimination by anti-Ig and C. This depletion of IgG₁ responses, assayed in adoptive transfer, is accompanied by increased responses of other isotypes, a phenomenon termed compensation. An analysis of the tertiary response reveals that cells sensitive to functional elimination are precursors to memory cells as well as plaque-forming cells. To investigate control mechanisms governing preferential expression of IgG₁, and the compensation active after elimination of IgG₁, increased numbers of carrier-primed cells were transferred and antigen dose was increased to adoptive recipients. Doses of carrier-primed cells or antigen were found which allowed maximal expression of both IgG₁ and non-IgG₁ isotypes. In recipients of anti-Ig and C-treated B cells, increased numbers of carrier-primed cells did not further increase the compensatory expression of non-IgG₁ isotypes. A discussion is presented of compensation as a result of limiting inductive signals to B cells.     

Vitamin B12 and the macromolecular composition of Euglena. I. Kinetic analysis of the cell cycle and chloroplast replication

The kinetics of cell division, chloroplast replication and mean DNA/cell of cultures progressing through B₁₂ deficiency do not follow smooth curves, but contain transient plateaus which are consistent mathematically with the hypothesis that one portion of the Euglena cell cycle, the S phase, is differentially extended under B₁₂ deficiency. A computer simulation of the B₁₂-deficient cultures was capable of duplicating the division kinetics of the actual culture. Chloroplast replication in B₁₂-deficient cells is not directly affected by B₁₂ deficiency, but is a function of the division kinetics of the cells. The chloroplasts continue to replicate initially at a high rate after the cells have entered B₁₂ deficiency, and then follow the kinetics of the cells.     

1H-N.M.R. spectra of glycosaminoglycan monomers and dimers in solution in methyl sulphoxide and water

¹H-N.m.r. spectra of glycosaminoglycuronan monomers and dimers in solution in methyl sulphoxide-d₆ have been investigated; N-H and O-H resonances were observed and partially assigned. Their temperature-dependence suggests hydrogen-bonding to the solvent, with the notable exception of that of HO-4 Of sodium D-gluctironate, which was consistently downfield and relatively temperature-insensitive. The concentration-dependence ofthis signal indicates that the corresponding hydroxyl group is involved in the formation of a dimer. Signals for N-H and O-H were observed for aqueous solutions, especially at subzero temperatures.     

Conformational analysis of glycosaminoglycans. I. Charge distributions, torsional potentials, and steric maps

An initial study of the charge distributions and torsional potentials for (1→4)-linked disaccharides, and a steric energy-map for (1→4)-linked disaccharides is reported for six acidic glycosaminoglycans. The charge distributions have been computed by the CNDO quantum-mechanical procedure, together with a molecular-decomposition technique. The intrinsic torsional potential has been computed by using CNDO energies and empirical potential-energies. The intrinsic torsional potential is two-dimensional. The general steric-map for a (1→4)-linked disaccharide will be useful for simplifying future conformational analyses of glycosaminoglycans. Wherever possible, comparisons between theory and experiment are presented or cited.     

Cholesterol autoxidation: identification of the volatile fragments.

The volatile fragments of air-aged cholesterol were analysed by means of gas chromatography-mass spectrometry; The following fourteen compounds were identified: ethanol, acetic acid, acetone, 2-methylpropene, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-butanone, 2-methylpropionic acid, 2-methyl-2-butanol, 2-pentanone, 3-methyl-2-butanone, 2-methyl-1-pentene, 2-methyl-2-pentanol, and 2-methyl-4-penten-2-ol. Their formation via decomposition of initially formed sterol hydroperoxides is discussed.     

Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Uptake and metabolism of purine nucleosides and nucleotides in isolated frog skeletal muscle.

When isolated frog skeletal muscles were incubated with ¹⁴C-labeled adenosine, the nucleoside was rapidly taken up by the cells and was either immediately incorporated into adenine nucleotides or deaminated to inosine. Incorporation was predominant at low (micromolar) concentrations whereas, deamination was the major route of metabolism at high (millimolar) concentrations. When muscles were incubated with ¹⁴C-labeled inosine the nucleoside, after entry into the cells, was metabolized to a lesser extent than adenosine. ATP and hypoxanthine were the major products of its metabolism. Intracellular concentrations were calculated using ³H-labeled sorbitol to measure the extracellular space.Because of its lower rate of intracellular metabolism inosine was used to investigate the characteristics of the nucleoside transport system. The uptake of inosine was saturable at high concentrations and was specifically inhibited by the presence of adenosine or uridine in the incubation media. Persantin, a well known specific inhibitor of nucleoside transport, also competitively inhibited inosine uptake, as did theophylline [1, Woo et al. Can J. Physiol. Pharmacol. $\text{52}$, 1063, 1974]. These data, along with the knowledge that in a well-oxygenated muscle, inosine entry follows a downhill chemical potential gradient, strongly support the view that the transport mechanism is facilitated diffusion.The muscle cell membrane does not appear to be permeable to ¹⁴C-labeled ATP under the conditions studied. Investigations of the permeability to the major extracellular degradation products of ATP suggest that AMP was the compound most likely to cross the cell membrane.     

The synthesis of radioactive 3-methylthiopropionate and other alkylthio fatty acids.

Aprocedure is described for the synthesis of radioactive 3-methylthiopropionate, a recently isolated metabolite of mammalian methionine metabolism. The method is a two-step synthesis whereby correspondingly labeled methionine is degraded by ninhydrin to 3-methylthiopropionaldehyde and then specifically oxidized to 3-methylthiopropionate without oxidation of the sulfur atom by the yeast enzyme, aldehyde dehydrogenase. Radiochemical purity of the isolated product was established by paper, thin-layer, and gas-liquid chromatography. This procedure is economical and readily applicable to the synthesis of other alkylthio fatty acids for the study of S-methylcysteine and ethionine metabolism and probably for the synthesis of radioactive intermediates of branched chain amino acids.     

Cholinephosphophotransferase activities in early rat embryos and their associated placentas.

CDP-Choline:1,2-diglycerolcholinephosphotransferase (EC 2.7.8.2, cholinephosphotransferase) activities were determined in subcellular fractions prepared from rat embryos, placentas, or yolk sacs obtained on the fourteenth day of gestation. It was found that, in all of the tissues studied, cholinephosphotransferase activity (1) copurified with NADPH-cytochrome c reductase activity (EC 1.6.2.4), (2) was maximal around pH 8.0; (3) was stimulated by MgCl₂, exogenous diolein, and cytidine diphosphocholine (CDP-choline); and (4) was highest in homogenates of placentas, lowest in those of embryos, and intermediate in those of yolk sacs. These data substantiate, for the first time, that the early mammalian (rat) embryo, placenta, and yolk sac have the ability to synthesize phospholipids de novo.     

A comparative study of mouse liver and mouse blastocyst DNA-dependent RNA polymerases.

DNA-dependent RNA polymerase has been studied in adult mouse liver and mouse blastocysts. The enzyme from mouse liver was resolved into three enzyme forms by DEAE-Sephadex chromatography. Two of the forms, IA and IB, are insensitive to α-amanitin, have low $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratios, and are optimally active at low ionic strength. Form II is inhibited by α-amanitin, has a higher $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratio, and is most active at high ionic strength. An optimal reaction temperature of 37 ° C was found for all enzyme forms. All of the isolated enzyme forms are inhibited by the exotoxin from Bacillus thuringiensis and the inhibition can be partially reversed by increased ATP levels. Forms IA and IB are most active with native template while form II prefers denatured DNA.The blastocyst RNA polymerase activity exhibits similar requirements for divalent metal ions and ionic strength to the purified liver enzymes. The maximum inhibition of blastocyst RNA polymerase obtained with α-amanitin and exotoxin differs from that observed for purified liver enzymes but is similar to the inhibition of liver homogenate. However, the concentrations of inhibitor required for maximum inhibition by α-amanitin and exotoxin is different for the blastocyst and liver homogenate enzymes.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

A new class of model glycolipids: synthesis, characterization, and interaction with lectins.

A method is described for preparing model glycolipids by linking aldobionic acids to an alkylamine through an amide bond. These compounds may be rapidly prepared in large quantities. The glycolipids precipitate specifically with lectins. Precipitation occurs at glycolipid concentrations just above their critical micelle concentration.     

Inhibition of hepatic gluconeogenesis and lipogenesis by benzoic acid,p-tert.-butylbenzoic acid,and a structurally related hypolipidemic agent SC-33459

Benzoic acid, p-tert.-butylbenzoic acid, and a structurally related hypolipidemic agent SC-33459 were found to inhibit glucose synthesis by hepatocytes isolated from 48-h fasted rats as well as fatty acid synthesis by hepatocytes isolated from meal-fed rats. Glucose synthesis was less sensitive than fatty acid synthesis. Benzoic acid was the least effective inhibitor of both processes; SC-33459 and p-tert.-butylbenzoic acid were very potent inhibitors with similar efficacy. Glycine prevented the inhibition of fatty acid synthesis caused by benzoic acid, but had no effect on that caused by p-tert.-butylbenzoic acid. Octanoate opposed the inhibitory effects of both benzoic acid and p-tert.-butylbenzoic acid. Oxidation of [1-¹⁴C]oleate to ketone bodies and acid-soluble radioactive products was inhibited by both p-tert.-butylbenzoic acid and SC-33459. Preincubation of hepatocytes with SC-33459 was required for the latter effect, suggesting catabolism of this compound may be involved. SC-33459 is a p-tert.-butylphenyl derivative which should be readily converted to p-tert.-butylbenzoic acid by β oxidation. Both p-tert.-butylbenzoic acid and SC-33459 decreased citrate levels dramatically. All three compounds reduced CoA and acetyl-CoA levels and increased medium-chain acyl-CoA ester levels. p-tert.-Butylbenzoic acid and SC-33459 also increased long-chain acyl-CoA ester levels. The increase in medium-chain acyl-CoA levels presumably reflects benzoyl-CoA formation from benzoic acid and p-tert.-butylbenzoyl-CoA formation from p-tert.-butylbenzoic acid and SC-33459. Inhibition of glucose and fatty acid synthesis by these compounds may be due to effects on specific enzymes or to CoA sequestration.     

Ornithine decarboxylase activity and DNA synthesis in Morris hepatomas 5123-C and 7800.

The activities of ornithine decarboxylase (ODC) and thymidine kinase (TK) and the rates of DNA synthesis were determined in hepatomas and livers of rats bearing Morris hepatoma 5123-C or 7800 and entrained to a schedule of 12 hours of light followed by 12 hours of darkness, with food (60% protein) available only during the first 2 hours of the dark period. ODC activity in hepatoma 5123-C displayed a diurnal oscillation, increasing 2-fold during the feeding period and then rapidly decaying to 20% of the peak level. The livers of rats bearing hepatoma 5123-C exhibited a similar oscillation of ODC activity, with peak values lower than in the hepatomas but higher than in the livers of control (non-tumor bearing) animals. TK activity and the rate of DNA synthesis in hepatoma 5123-C were low during most of the dark period but increased rapidly towards the end of the dark period. DNA synthesis reached a plateau at the dark-light interface and then rapidly declined, but TK activity remained high during the light period. Similar studies on hepatoma 7800 established that ODC activity in this hepatoma did not oscillate but remained at low levels throughout the day. Similarly, host livers of rats bearing hepatoma 7800 did not exhibit the diurnal oscillation of ODC activity characteristic of liver from control rats, but showed a slow increase in activity followed by a plateau and a slow decline to base-line levels. DNA synthesis in hepatoma 7800 was constant throughout the day, whereas TK activity may have increased during the dark period. In the livers of control rats and animals bearing hepatoma 5123-C or 7800, TK activity and rate of DNA synthesis were at low levels at all times studied and appeared not to oscillate.     

Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr ⁵¹Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.     

In vitro and in vivo induction of effector T cells mediating DTH responses to a protein and a synthetic polypeptide antigen.

We have developed an in vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl γ-globulin (FγG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro- and in vivo-activated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty × 10⁶ “educated” cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.