Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Electrocatalytic oxidation of guanine, guanosine, and guanosine monophosphate

The electrochemical behavior of guanine, guanosine, and guanosine monophosphate (GMP) at redox polymer film modified indium tin oxide electrodes is examined by voltammetry and redox titration. Utilizing the redox polymer-coated electrodes as indicator electrodes, a new method for measuring the oxidation potentials, based on monitoring their catalytic oxidation by different redox polymer coated electrodes at different pH, was proposed in this work. The oxidation potentials of 0.81 V and 1.02 V versus normal hydrogen electrode were determined for guanine and guanosine/GMP under physiological conditions, the lowest oxidation potentials ever reported, to our knowledge.     

Transport of cyclic adenosine 3'',5''-monophosphate across Escherichia coli vesicle membranes.

The uptake and efflux of cyclic adenosine 3',5'-monophosphate (3',5'-cAMP) by Escherichia coli membrane vesicles were studied. Metabolic energy was not required for the uptake process and was found to actually decrease the amount of 3',5'-cAMP found in the vesicles. 3',5'-cAMP uptake exhibits saturation kinetics (Km = 10 mM, Vmax = 2.8 nmol/mg of protein per min) and was competitively inhibited by a number of 3',5'-cAMP analogs. The uptake of 3',5'-cAMP was found to be sharply affected by a membrane phase transition. The excretion of 3',5'-cAMP was studied by using everted membrane vesicles. Efflux in this system was dependent upon metabolic energy and was reduced or abolished by uncouplers. Different energy sources powered efflux at different rates, showing a relationship between the degree of membrane energization and rate of excretion of 3',5'-cAMP. The efflux process also displayed saturation kinetics (Km = 10.0 mM, Vmax = 0.98 nmol/mg of protein per min) and was competitively inhibited by the same 3',5'-cAMP analogs and to the same degree as was the uptake process. 3',5'-cAMP was found to be chemically unaltered by both the uptake and excretion processes. These data are interpreted as showing that the uptake and excretion of 3',5'-cAMP in E. coli membrane vesicles are carrier-mediated phenomena, possibly employing the same carrier system. Uptake is by facilitated diffusion whereas efflux is via an energy-dependent, active transport process. Evidence is presented showing that cells can regulate the number of 3',5'-cAMP transport carriers. The rate of 3',5'-cAMP excretion is possibly regulated by both the degree of membrane energization and the number of carriers present per cells.     

A simple radioimmunoassay for 3',5', cyclic adenosine monophosphate.

A radioimmunoassay for 3′,5′ cAMP has been developed in which [³H]3′,5′ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is 0.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotides. Samples are prepared by ethanol precipitation. Tissue levels of 3′,5′ cAMP are comparable to those reported by others.     

Interaction of duramycin with artificial and natural membranes

Duramycin is a polypeptide antibiotic (molecular weight 2012) obtained from culture filtrates of Streptomyces cinnamomeus forma azacoluta. In this work, we show that low concentrations of duramycin induced aggregation of lipid vesicles containing unsaturated phosphatidylethanolamine and unsaturated monogalactosyl diglyceride, and of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Furthermore, duramycin inhibited the ATP-dependent Ca2+ uptake in sarcoplasmic reticulum vesicles without affecting the hydrolysis of ATP or the permeability of Ca2+. Also, duramycin only inhibited the bacteriorhodopsin proton pump reconstituted into phospholipid vesicles containing phosphatidylethanolamine. We have isolated a duramycin-resistant strain of Bacillus subtilis and have mapped the location of duramycin resistance. In this strain, the secretion of protons and influx of calcium were resistant to duramycin, and its lipid composition was profoundly different from that of the parent strain. No phosphatidylethanolamine was detected in the resistant strain. Our findings are consistent with the idea that duramycin recognizes a particular membrane conformation determined by the presence of phosphatidylethanolamine or monogalactosyl diglyceride.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae

We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system. The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool. However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally. This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.     

Light-initiated changes of cyclic guanosine monophosphate levels in the frog retina measured with quick-freezing techniques

Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.     

The cyclic adenosine monophosphate phosphodiesterases of myoblasts, fibroblasts, and their somatic cell hybrids.

Three forms of cAMP phosphodiesterases are found in mouse L cells (fibroblasts) and rat skeletal myoblasts. The myoblast enzymes can be resolved by chromatography on DEAE-cellulose and the fibroblast enzymes by chromatography on DEAE-Biogel. The myoblast enzymes are "high affinity" cAMP specific forms and have different molecular weights, while all L-cell enzymes have an apparent molecular weight of 450,000. Only one of the L-cell enzymes is able to hydrolyze both cyclic guanosine monophosphate (cGMP) and cAMP. Hydrolysis of the latter is stimulated by micromolar amounts of cGMP. The myoblast x L cell hybrids possess at least five phosphodiesterases, three of which can be identified as being of myoblast or fibroblast origin. One of the fibroblast enzymes appears to be modified in hybrids. The entire phosphodiesterase regulatory system of the myoblasts is active in the hybrids.     

Efflux of cyclic adenosine monophosphate from cells: mechanisms and physiological implications

Although cyclic nucleotides are hydrophilic compounds, extracellular cAMP (cAMPo) rapidly accumulates during the activation of adenylate cyclase. This review considers the kinetic characteristics of cAMP transport through the plasma membrane and its physiological implications. The influx and efflux of cAMP occur via different carriers. At physiological concentrations of cAMPo, the influx of cAMP does not significantly contribute to regulation of the intracellular content of the cyclic nucleotide, but it is responsible for the accumulation of cAMPi in experiments at [cAMP]o approximately 1 mM. In contrast, the high rate of cAMP efflux is mainly responsible for normalization of [cAMP]i during long-term activation of adenylate cyclase. The possible involvement of ATP-binding cassette proteins (ABC proteins) in the efflux of cAMP from the cell is considered. In procaryotes cAMPo is a signal molecule during the generation of cell colonies, acting on special receptors that interact with GTP-binding proteins. Such receptors have not been found in vertebrates, and in most cases the signal functions of cAMPo are mediated by its degradation by extracellular enzymes with subsequent activation of adenosine receptors.     

Correlated effects of acetylcholine and cyclic guanosine monophosphate on membrane properties of mammalian neocortical neurons

The effects of successive extracellular iontophoresis of acetylcholine (ACh) and atropine, and intracellular hyperpolarizing iontophoresis of cyclic GMP (cGMP) were studied in single neurons of the coronal-pericruciate cortex of awake cats. (a) Fifty-seven percent of the neurons that were tested responded to ACh with an increase in neuronal input resistance (Rm) and 50% responded to ACh with an increase in firing rate; 65% responded to cGMP with an increase in Rm and 60% responded to cGMP with an increase in firing rate. (b) After application of atropine, increases in Rm and firing rate associated with iontophoresis of ACh failed to recur. (c) Persistent increases in Rm following application of ACh accompanied by current-induced neuronal discharge were not diminished by subsequent application of atropine. (d) Atropine did not prevent increases in Rm and firing rate associated with intracellular iontophoresis of cGMP. (e) All cells tested with both ACh and cGMP that were shown initially to respond to extracellular ACh with increases in Rm were later shown to have comparable responses to cGMP.     

Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many ³²P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-³²P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-³²P] phosphate from [γ-³²P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]₀ e^?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [³²P] phosphate from [γ-³²P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.