Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.     

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.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. IV. Evidence for participation of prostaglandins of the E series in the early events.

The addition of KLH to KLH-primed rabbit lymph node cell cultures induced an anamnestic antibody response. The further addition of prostaglandins of the E series, but not PGF1^α, enhanced this antibody response manifold. The addition to these cultures of prostaglandin synthetase inhibitors together with KLH inhibited antibody production. At the concentration (10^?4) required to inhibit antibody synthesis, by a variety of criteria one of these inhibitors, indomethacin, was shown not to exert its effects through cytotoxicity. By contrast, two other inhibitors of prostaglandin synthesis, Ro-20-5720 and Ro-3-1314, inhibited antibody synthesis because of their cytotoxicity. The inhibition of the antibody response by indomethacin did not occur when PGE1 or PGE2 was added concurrently to these cultures, clearly showing that inhibition was due to a deficiency of prostaglandins. These findings strongly suggest that induction and/or regulation of the in vitro anamnestic antibody response of KLH-primed lymph node cells to 1 and 100 μg KLH requires continued prostaglandin synthesis. Potential mechanisms for the regulation of the antibody response by prostaglandins are discussed.     

Cellular levels of the prophage lambda and 434 repressors.

As a prerequisite to a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels.Using the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to the binding mixture; this procedure was without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied with the method of cell disruption.The cellular concentration of λ repressor, about 140 active repressor molecules per monolysogen, was relatively constant under specific cultural conditions. The repressor concentration increased with the number of cI gene copies but not in direct proportion.The 434 repressor concentration, hardly detectable in extracts of lysogens carrying an imm434 prophage, was greatly enhanced in bacteria carrying the newly constructed plasmid pGY101, that encodes the 434 cI gene.The cellular repressor level produced by 434 is lower than that produced by λ: this indicates that the maintenance of the prophage state is ensured by a relatively small number of repressor molecules binding tightly to the operator sites.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.     

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.     

Preincubation stimulates fatty acid synthesis by liver slices and isolated hypatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-¹⁴C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10^?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-¹⁴C] acetate into fatty acids or cholesterol. The rate of ¹⁴CO₂ synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-¹⁴C] acetate or [³H] H₂O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control     

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.     

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.     

An automated differential scanning dilatometer.

A sensitive scanning dilatometer has been constructed which continuously records the apparent partial specific volume of small biological samples (10–100 mg) as a function of temperature. Changes in volume of 0.02 μl can be resolved. Tedious calibrations with their inherent errors are eliminated by employing a differential approach in which a recording electrobalance responds to the difference in buoyancy of identical sample and reference tubes. Agreement with the literature values for the specific volume of n-eicosane and the apparent partial specific volume of KCl was excellent. Apparent partial specific volumes obtained for dipalmitoyl lecithin, egg albumin, and bovine serum albumin agreed well with published values.     

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.