Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

Detection of two forms of glutamine synthetase in Bacillus brevis (A. G. 4)

A laboratory isolate of $\text{Bacillus}\text{brevis}$ could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg⁺⁺ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.     

The disappearance rate of intraventricular bradykinin in the brain of the conscious rat

To explore the possibility that cyclic nucleotides control green algal growth and division, $\text{Chlamydomonas}$ chemostat cultures were assayed for cyclic nucleotides. Substantial qualities of cAMP were found in cells and in extracellular millieu. Most cGMP molecules were extracellular. Slowing cell growth by slowing chemostat dilution caused reversible changes in cellular morphology and cyclic nucleotide levels. During slowed growth cAMP level increased dramatically; cGMP level decreased. New cells resulting from division were not released from original cell wall.     

Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1?2}\text{s}$) and a class of slow-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15?150}\text{s}$). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase.     

Dibutyryl cGMP: inhibitor of the effect of cholecystokinin and gastrin on the guinea pig gallbladder in vitro

N², O²-di-butyryl guanosine 3′:5′ monophosphate (Bt₂ cGMP), a known competitive and selective inhibitor of the effect of cholecystokinin on the pancreatic acinar cells $\text{in}$$\text{vitro}$ was tested for its effect on the guinea pig gallbladder $\text{in}$$\text{vitro}$. Bt₂ cGMP inhibited competitively the contractile effect of cholecystokinin octapeptide, and also inhibited the contraction induced by sulfated gastrin-17. Bt₂ cGMP failed to inhibit the contraction induced by bombesin, acetylcholine or histamine. The 8-bromo derivative of cGMP and the dibutyryl derivative of cAMP did not affect contraction stimulated by cholecystokinin octapeptide. Since it is specific for gastrincholecystokinin peptides, and not restricted to the pancreas, Bt₂ cGMP could be used to recognize the action of these peptides.     

Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa.

The effect of the nitrogen and carbon sources in the regulation of g$\text{l}$u tamine synthetase has been studied in fed-batch cultures of $\text{Neurospora crassa}$. The limitation of ammonium in an excess of the carbon source, leads to an accumulation of α-ketoglutarate and elevation of glutamine sy$\text{n}$ thetase. The limitation of sucrose in an excess of ammonium results in a decrease in glutamine synthetase activity. These results indicate that the carbon source exerts a positive control in the regulation of glutamine synthetase.     

Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

Studies were conducted to determine whether normal and/or neo-plastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the $\text{in}$$\text{vitro}$ addition of 10 μM DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 μM DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 μM BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, α-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.     

Cyclic adenosine 3':5'-monophosphate levels in normal and transformed cells of higher plants

Cyclic adenosine 3′:5′-monophosphate (cAMP) concentrations were determined for various normal and transformed (crown-gall) plant tissues grown in sterile culture. No significant differences in cAMP concentrations were found between normal and transformed cells of $\text{Vinca rosea, Helianthus annuus}$, and $\text{Nicotiana tabacum}$, unlike the suppressed synthesis observed in transformed cells of mammalian systems. cAMP concentrations of these tissues in culture averaged 135 nanomolar. No correlation was found between cAMP concentrations and tissue culture generation times.     

An unusual cyclic AMP-dependent protein kinase from nematodes

The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode $\text{Aphelenchus}$$\text{avenae}$. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the $\text{cAMP}\text{cGMP}$ kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.     

Dissociation by elastase digestion of enzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver

Glutamine-dependent carbamyl phosphate synthetase of rat liver, purified about 2,100-fold, existed as a complex with aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, with a sedimentation coefficient of 27 S. Treatment of this complex with pancreatic elastase caused a selective inactivation of the transcarbamylase with concomitant dissociation of the complex. The dissociated synthetase was as sensitive to allosteric effectors as the enzyme within the complex, but had a 5 times higher apparent $\text{Km}$ for MgATP^2?. This change appears to be intimately related to the release of the enzyme from the complex.     

Biosynthesis of beta-phenethyl alcohol in Candida guilliermondii.

$\text{Candida guilliermondii}$ produced β-phenethyl alcohol and β-phenyllactic acid when grown in a synthetic medium containing L-phenylalanine as sole source of nitrogen. The cell-free preparations from these cells showed the following enzymes: phenylalanine aminotransferase, phenylpyruvate decarboxylase, phenylpyruvate reductase and phenylacetaldehyde reductase. The cell-free preparations of $\text{C. guilliermondii}$ grown in medium with ammonium sulfate, lacked these enzyme activities, indicating the inducible nature of these enzymes. The results indicate the role of β-phenylpyruvate as a key intermediate in the pathway of biosynthesis of β-phenethyl alcohol and β-phenyllactic acid from L-phenylalanine.     

Resting and concanavalin-A stimulated levels of cyclic nucleotides in splenic cells of aging mice with spontaneous cancers.

The resting levels of cyclic 3′, 5′ -adenosine monophosphate (cAMP) and cyclic 3′, 5′ -guanosine monophosphate (cGMP) in splenic lymphoid cells of 25 aged (C57BL/10 × C3H)F₁ hybrid mice with spontaneous tumors, including 5 with hepatoma, 10 with lung tumor, 2 with lymphoma, and 8 with several varieties of tumor, as well as in 18 young and 13 tumor-free aging mice, were measured. The alterations in cyclic nucleotide levels in spleen cells characteristic of normal aging in tumor-free animals may be additionally influenced by the occurrence of spontaneous neoplasia. Furthermore, the levels may vary with different types of late-life tumors. For example, levels of cAMP in resting spleen cells of old mice with hepatomas were not different than in age-matched controls, whereas spleen, cells from old mice with lung tumors showed exceedingly high levels of resting cAMP. Upon $\text{in vitro}$ stimulation by Con-A, the splenic lymphoid cells from mice bearing spontaneous late-life lung and liver tumors displayed different kinetic patterns of percent changes in cAMP, cGMP and cAMP/cGMP ratios when compared to either young or age-matched tumor-free controls. Thus, both resting and Con-A stimulated levels of cAMP and cGMP and their ratios in splenic lymphoid cells may be affected by spontaneous cancer elsewhere in the body, including cancer of non-lymphoid type and origin. These findings plus the known functional decline in immune response capacity and the increase in spontaneous tumor incidence with age may suggest the existence of a complex relationship among cyclic nucleotide levels, immunity, aging, and cancer.     

Simian virus 40 rapidly lowers cAMP levels in mouse cells

The addition of SV40 to contact inhibited $\text{Balb}\text{3T3}$ cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.     

On the mechanism of tolerance to isoproterenol-induced accumulation of cAMP in rat pineal in vivo.

Less cyclic adenosine 3′:5′ monophosphate (cAMP) accumulated in rat pineal gland, $\text{in}$$\text{vivo}$, after two doses of l-isoproterenol (5mg/kg, i.p.) than after one dose. A single injection of l-isoproterenol decreased the ability of l-isoproterenol to activate adenylate cyclase and increased the activity of the low Km phosphodiesterase (PDE). Tolerance to l-isoproterenol-induced accumulation of cAMP in rat pineal $\text{in}$$\text{vivo}$ may be due to decreased responsiveness of adenylate cyclase as well as to increased activity of PDE.     

Coenzyme A: fatty acid synthetase apoenzyme 4'-phosphopantetheine transferase in yeast

A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined $\text{in}$$\text{vitro}$ to form a hybrid apoenzyme complex. $\text{In}$$\text{vivo}$ the corresponding $\text{Saccharomyces}$$\text{cerevisiae}$$\text{fas}$-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed $\text{in}$$\text{vitro}$ could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.     

Enxymology of human colon tumors

The biochemical strategy of human colon adenocarcinoma was studied by elucidating the enzymic programs of pyrimidine biosynthesis and degradation, glycolysis, pentose phosphate production, and galactose metabolism in normal colon mucosa and in 9 cases of primary colon adenocarcinoma. Enzymic activities were determined in the 100,000 X $\text{g}$ supernatant fluid with spectrophotometric or isotopic assays under optimum conditions yielding linear kinetics. In the human colon tumors the activities of enzymes of the $\text{de}$$\text{novo}$ pyrimidine biosynthesis, CTP synthetase, OMP decarboxylase, and orotate phosphoribosyltransferase, were increased to 348, 183, and 201% of those of normal human colon mucosa. The activities of the salvage pathway enzymes, thymidine kinase, uracil phosphoribosyltransferase and uridine kinase, were increased to 331, 254 and 281%. By contrast, the activity of the catabolic enzyme, uridine phosphorylase, was decreased to 69%. The ratio of activities of uridine kinase/ uridine phosphorylase was elevated to 564%. The activities of the key glycolytic enzymes, hexokinase and pyruvate kinase, and those of pentose phosphate production, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transaldolase, increased to 348, 209, 262, 156, and 180% respectively. The activity of the first committed enzyme of galactose utilization, galactokinase, was increased to 315%. The enzymic program of human primary colonic adenocarcinoma was similar in most respects to that which we observed in chemically-induced, transplantable adenocarcinomas of the colon in mouse and in rat (4). The reprogramming of gene expression in human colon tumor provides an increased capacity for biosynthesis of pyrimidines and ribose 5-phosphate, and for utilization of the glycolytic pathway and of galactose. These alterations in gene expression should confer selective advantages to the human colon tumor cells. The marked elevations in the activities of the salvage enzymes, uridine-cytidine kinase and thymidine kinase, explain in part the failure to obtain good therapeutic results with inhibitors of the $\text{de}$$\text{novo}$ pathway and account, in part at least, for the clinical difficulties encountered in the treatment of colon tumors. The elevated activities of CTP synthetase, OMP decarboxylase, uridine-cytidine kinase and thymidine kinase mark out these enzymes as targets for combination chemotherapy. Through such enzyme-pattern-targeted chemotherapy the drug treatment of human colon tumors should be improved.     

Glutamine-requiring mutants of Bacillus subtilis.

Two glutamine-requiring (Gln^?) mutants of $\text{Bacillus subtilis}$ SMY were deficient in glutamine synthetase activity $\text{in vitro}$. The Gln^? mutants sporulated poorly unless glutamine was provided at high concentrations. The differential rate of histidase synthesis following induction was 4- to 6-fold higher in the Gln^? mutants than in wild-type cells. In addition, glucose repression of utilization of alternative carbohydrates appeared to be partially relieved in the Gln^? mutants.     

Effect of oxygen tension on nitrogenase and on glutamine synthetases I and II in Rhizobium jaonicum 61A76.

When grown under aerobic conditions, $\text{Rhizobium japonicum}$ 61A76 contains two forms of glutamine synthetase, GSI and GSII, as previously described. In contrast, cells grown under the low O₂ tensions required for nitrogenase synthesis contain only GSI. GSII activity disappears completely at O₂ levels below 0.4%. GSI activity decreases by only 50%, but the enzyme appears to become highly adenylylated under the low O₂ tensions required for nitrogenase synthesis.