Assays for Intracellular Cyclic Adenosine Monophosphate (cAMP) and Lysosomal Acidification.

Cyclic adenosine monophosphate (3',5'-cAMP) is a multifunctional second messenger which controls extremely diverse and physiologically important biochemical pathways. Among its myriad roles, 3',5'-cAMP functions as an intracellular regulator of lysosomal pH, which is essential for the activity of acidic lysosomal enzymes. Defects in lysosomal acidification are attributed to many diseases like macular degeneration, Parkinson's, Alzheimer's, and cystic fibrosis. Strategic re-acidification of defective lysosomes by pharmacological increase of intracellular cAMP offers exciting therapeutic potential in these diseases. Modular assays for accurate assessment of intracellular cAMP and lysosomal pH are a critical component of this research. We describe label-free targeted metabolomics for quantitating intracellular cAMP and integrated assays for measuring lysosomal pH. These hybrid assays offer fast, unbiased information on intracellular cAMP concentrations and lysosomal pH that can be applied to many cell types and putative drug screening strategies.

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules.

BACKGROUND: Pathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa. RESULTS: A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid-virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets. CONCLUSIONS: VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.

RNA aptamer based electrochemical biosensor for sensitive and selective detection of cAMP.

Cyclic adenosine monophosphate (cAMP) is an important small biological molecule associated with the healthy state of living organism. In order to realize highly sensitive and specific detection of cAMP, here an RNA aptamer and electrochemical impedance spectroscopy (EIS) based biosensor enhanced by gold nanoparticles electrodeposited on the surface of gold electrode is designed. The designed aptasensor has a wide effective measuring range from 50pM to 250pM with a detection limit of 50pM in PBS buffer, and an effective measuring range from 50nM to 1µM with a detection limit of 50nM in serum. The designed biosensor is also able to detect cAMP with high sensitivity, specificity, and stability. Since the biosensor can be easily fabricated with low cost and repeatedly used for at least two times, it owns great potential in wide application fields such as clinical test and food inspection, etc.

[Extract process of cyclic adenosinemonophosphate (cAMP) in Ziziphus jujuba].

OBJECTIVE: To study the extract process of Cyclic adenosinemonophosphate (cAMP) in Ziziphus jujuba. METHODS: CAMP was extracted with water, separated and purificated by ion exchange and silica gel G column chromatography. RESULTS: Thin layer chromatography (TLC) of the extracts and control cAMP showed the same Rf 0.75; The UV-absorption of the extracts and control cAMP had maximal absorb peak at 260 nm and the least absorb value at 230 nm; Infra-red spectrum of the extracts was indistinguishable from that of control cAMP; Retention time of the extracts by high-performance liquid chromatography (HPLC) determinate was 5.237 minute,unsettled impurity peak,content was 97%. Control cAMP was 5.350 minute,their retention time were of little difference. CONCLUSION: Chemistry structure of this extract with that of control cAMP is the same.

Cyclic nucleotide content of tobacco BY-2 cells.

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.

Measurement of adenosine 3',5'-cyclic monophosphate and guanosine 3', 5'-cyclic monophosphate in mussel (Mytilus galloprovincialis lmk.) by high-performance liquid chromatography with diode array detection.

Two fast and sensitive methods for the determination of cAMP and cGMP levels in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. are described. Both methods use ion-pair high-performance liquid chromatography with diode array detection. The use of the diode array detector permitted the simultaneous detection of the absorbance at two different wavelengths and the obtaining of the UV absorption spectrum for each detected peak, confirming peak purity and identity. Method precision was good. The detection limit for both nucleotides was 2.5 pmol (signal-to-noise ratio = 4 at 254 nm). Previous to the injection onto the chromatograph, both nucleotides were purified by precipitation of the adenine and guanine 5'-ribonucleotides with ZnSO(4)-Na(2)CO(3). The supernatant obtained was subsequently passed over an anion-exchange column (AG l-X8 formate form resin). Early results seem to indicate that there is a seasonal variation in the contents of both cyclic nucleotides in mantle tissue. Such variation is probably related to the annual gametogenic cycle.

Age-related loss of immunoregulatory function in peripheral blood CD8 T cells.

CD8+ T cells from young individuals become inhibitory for the (Staphylococcus aureus + interleukin 2)-induced differentiation of autologous B cells into immunoglobulin secreting cells (ISC) after exposure to pokeweed mitogen (PWM), dimaprit or intracellular cAMP raising agents, such as forskolin or dibutyryl-cAMP. In the present study this immunoregulatory activity was found to be lacking in CD8+ T cells from peripheral blood lymphocytes (PBL) of aged (> 67 years old) subjects. Splenic CD8+ T cells from most individuals examined, including some aged subjects, exhibited this activity. While an age-related decrease in the CD8+ T cell subset, primarily in the virgin CD8+ T cells in PBL, was detected, this decrease was not sufficient to explain a total absence of activity. There was no age-related decrease in cAMP upregulation by forskolin or dimaprit in peripheral blood T cells. However, whereas PWM induced a highly significant increase in mRNA for transforming growth factor-beta (TGF-beta) in T cells from young individuals, no such increase could be detected in T cells from aged subjects. It is suggested that the decrease in immunoregulatory activity in PBL from the elderly may at least in part be due to a decrease in TGF-beta production.

Mediation of renal cyst formation by hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent mitogen for renal tubular cells, and HGF and its receptor (c-met proto-oncogene product, Met) can induce lumen formation in epithelial cells. We measured the concentration of HGF in cyst fluids from patients with renal cystic diseases. The concentration of HGF was high in proximal cyst fluid (mean 2.45 vs 0.42 ng/mL in distal cysts). Cyclic AMP concentration was higher in distal than in proximal cysts (663 vs 6.0 pmol/L). mRNA of HGF and Met were co-expressed in cyst walls from cases with polycystic kidney disease. These findings suggest that HGF is a growth factor that may mediate human renal cyst genesis.

Identification of 3',5'-cyclic AMP in axenic cultures of Lemna paucicostata by high-performance liquid chromatography.

Nucleotides were purified from the extract of aseptically grown Lemna paucicostata 6746 by low-pressure column chromatography and thin-layer chromatography. In this partially purified sample, a compound has been identified, using a highly sensitive HPLC-fluorometric detection method, with chromatographic properties and substrate specificity for cyclic nucleotide phosphodiesterase similar to authentic 3',5'-cyclic AMP. The level of cAMP has been estimated to be 70-80 pmol/g fresh wt.

In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes.

The promoter region preceding the hutUH operon in Klebsiella aerogenes contains two oppositely oriented, overlapping promoters. In the absence of catabolite gene activator protein-cyclic AMP (CAP-cAMP), transcription proceeds primarily from the backward-oriented promoter (Pc), whose function has not yet been determined, and only very weakly from the forward hutUH promoter, hutUp. In the presence of CAP-cAMP, Pc is repressed and transcription from hutUp is favored. Two protein components required for this in vitro transcription system, RNA polymerase (RNAP) and CAP, were purified from K. aerogenes and were shown to be functionally interchangeable with the corresponding proteins from Escherichia coli, suggesting that E. coli RNAP could be used to study some aspects of hut transcription. We showed that a gradual activation of hutUp (by increasing concentrations of CAP, cAMP, or glycerol) resulted in a parallel repression of Pc, arguing in favor of a direct competition between the two promoters. The presence of a DNA sequence resembling the consensus for CAP-binding sites and centered at nucleotide -82 (relative to hutUp) initially suggested that a primary role of CAP was to repress Pc, thereby indirectly activating hutUp. However, the relatively slow formation of open complexes at Pc, even in the absence of CAP-cAMP, showed that Pc is a weak promoter and likely to be a poor competitor for RNAP. The observed dominance of Pc over hutUp suggested that the latter is an even weaker promoter. Thus, repression of Pc would not be sufficient to cause the observed increase in hutUp activity, and the CAP-cAMP complex must play a direct role in the activation of hutUp.

A single column method for the assay of adenylate cyclase.

An improved, one-step method for the separation of cyclic AMP from other nucleotides on disposable columns of neutral aluminum oxide is described. The method consists of several modifications of an established assay for adenylate cyclase. These modifications were designed to increase the sensitivity of the method, to decrease the time required for column preparation, and to eliminate the variable elution patterns for cyclic AMP that are obtained when using aluminum oxide from different commercial sources. Uniform elution patterns and high recoveries (approximately 80%) of cyclic AMP were obtained when 0.1 M ammonium acetate was used to elute cyclic AMP instead of Tris-HCl buffer. Prior to column chromatography, the adenylate cyclase reactions were terminated with the addition of hydrochloric acid and the mixtures were heated to degrade acid-labile nucleotides that would otherwise elute with cyclic AMP from aluminum oxide columns. Disposable polypropylene columns, fabricated with a reservoir and fast-flow filters, were used for column chromatography. Low blank values, generally less than 15 dpm/assay tube, were obtained when the acidified reaction mixtures were applied directly to aluminum oxide columns without prior neutralization. The proposed method should be useful for the routine assay of adenylate cyclase activity.

[Cyclic nucleotides in a high molecular weight aminoacyl-tRNA synthetase complex from the liver of normal and irradiated rats]. / Tsyklichni nukleotydy u skladi vysokomolekyliarnogo kompleksu aminoatsyl-tRNK-syntetaz iz pechinky normal'nykh ta oprominenykh shchuriv.

The content of cAMP and cGMP in the composition of the high-molecular complex aminoacyl-tRNA-synthetase from the rat liver has been studied. The ionizing radiation decreases the level of the cyclic nucleotides in the composition of the complex. A problem on interregulation of the cyclic nucleotides and prostaglandins in the complex has been examined.

A rapid and economical method of preparing radioiodinated cyclic nucleotide derivatives for use in radioimmunoassays.

2'-O-succinyladenosine 3':5'-cyclic monophosphate tyrosyl methyl ester (ScAMP-TME) and 2'-O-succinylguanosine 3':5'-cyclic monophosphate tyrosyl methyl ester (ScGMP-TME) were radioiodinated using chloramine T and Na125I. The resulting radiolabeled cyclic nucleotide derivatives, ScAMP-125I-TME and ScGMP-125I-TME, were subsequently purified by reverse-phase chromatography on Sep-Pak C18 cartridges (Waters Associates, Milford, MA) and tested as tracers in sensitive radioimmunoassays for cAMP and cGMP, respectively. Purified ScAMP-125I-TME and ScGMP-125I-TME functioned in the respective radioimmunoassays for up to 12 weeks when suspended in a 1:1 (v:v) mixture of n-propanol and 20 mM sodium acetate, pH 6.0. Thus, this purification method enables rapid and economical preparation of tracers for cyclic nucleotide radioimmunoassays. Furthermore, our findings suggest that reverse-phase chromatography may be applicable to the purification of other small polar molecules to which tyrosyl groups have been added for the purpose of radioiodination.

The adenylate cyclase response to parathyroid hormone in cultured rabbit marrow fibroblastic cells.

The ability of fibroblastic cells to respond to parathyroid hormone (PTH) by an increase in adenylate cyclase activity is accepted as a characteristic of the osteogenic phenotype. Whether marrow fibroblastic cells, which have osteogenic potential when assayed in vivo, demonstrate this hormonal response when cultured in vitro has been investigated. Our study has shown a level of stimulation of adenylate cyclase activity by PTH in cultured rabbit marrow fibroblasts comparable with other osteogenic cells in vitro. The effect is seen in fibroblasts grown either from multiple colonies or from single colonies. Only a proportion of colonies had osteogenic potential in vivo assay and our results show a similar finding for the PTH response in vitro. To what degree the two parameters are expressed by the same colony has not yet been established.

Purification of 125I-labeled succinyl cyclic nucleotide tyrosine methyl esters by high-performance liquid chromatography.

Carrier free 125I-labeled succinyl cyclic adenosine monophosphate (ScAMP) and succinyl cyclic guanosine monophosphate (ScGMP) tyrosine methyl esters (TME) were purified by reversed phase high-performance liquid chromatography (HPLC) or descending paper chromatography. Using an isocratic buffer for HPLC, mono-ScAMP-125I-TME and mono-ScGMP-125I-TME were eluted from a C18 column at 8.9 and 6.9 min, respectively. Both of the mono-iodinated radioligands were completely separated from their noniodinated precursors and other iodinated products. The radioligands purified by HPLC or paper chromatography were used for the radioimmunoassay (RIA) of cAMP and cGMP. Cyclic AMP or cGMP inhibited binding of the HPLC purified radioligands at three- to fivefold lower concentrations than the paper chromatography purified radioligands. The sensitivity of the RIA decreased with time if paper chromatography purified radioligands were used, but remained stable for 4 months if the HPLC purified compounds were used, even with storage at 4 degrees C. We attribute these results to better purification of radioligands by the HPLC than by the paper chromatography. Using optimal conditions the HPLC method takes only 10 min and results in a high yield (greater than 95%) of added 125I into the monoiodinated products.

Timolol inhibits adenylate cyclase activity in the iris-ciliary body and trabecular meshwork of the eye and blocks activation of the enzyme by salbutamol.

The enzymatic activity of adenylate cyclase in homogenates and membrane-rich fractions prepared from rabbit iris-ciliary bodies and bovine trabecular meshwork was found to be inhibited by timolol. Treatment of iris-ciliary body homogenates with Triton X-305 resulted in abolition of the inhibitory effect of the drug on the activity of the enzyme. The stimulatory effect of salbutamol on the enzyme was also susceptible to blockade by timolol. It is suggested that the hypotensive action of timolol on intraocular pressure results from structural and functional changes induced on the plasma membranes of the iris-ciliary body and trabecular meshwork by the thiadiazole group of the molecule, and, also, from the occupation of the adrenergic receptors of the iris-ciliary body by the tert-butylamino-2-hydroxypropoxy part of the compound.