The assay of adenosine 3':5'-cyclic monophosphate by aturation analysis.
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Adenine Nucleotides↗
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Simultaneous analysis of adenosine 3',5'-cyclic monophosphate accumulation and adenosine 5'-triphosphate metabolism in cultured cells preincubated with [2-3H]adenine.
A simple and sensitive method based on metabolic labeling was developed for the simultaneous analysis of cyclic AMP accumulation and ATP metabolism in small numbers of cultured cells. Cells are preincubated overnight with [2-3H]adenine to label the ATP pool to a high specific activity. After cell stimulation the metabolites are extracted in a small volume of aqueous acetic acid and chloroform and separated without further manipulation by one-dimensional thin-layer chromatography and the radioactivity incorporated is determined by liquid scintillation counting. With ATP labeled to about 6 Ci/mmol, the lower limit of cyclic AMP detection is 2 fmol, a sensitivity that is comparable to the radioimmunoassay of acetylated cyclic AMP. In primary neurons and a neural cell line, for example, levels of ATP and its metabolites change when large amounts of cyclic AMP are generated, each with its unique pattern. ATP is also depleted when metabolic energy is consumed concomitantly with stimulation of cyclic AMP production by agonists, probably as a result of an increase in ion pump activity following cation influx. As ATP is utilized for cyclic AMP production and simultaneously for many other processes, an assessment of its metabolism in parallel with that of cyclic AMP is critical. We suggest that the method described here is particularly advantageous over other methods for this purpose.
Adenine↗
Cyclic flow-injection analysis for the repetitive determination of zinc with 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol and EDTA.
A circulatory flow-injection method (cyclic FIA) for the repetitive determination of zinc has been proposed. The procedure involves the use of 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS) together with EDTA as a reagent carrier solution, which is recycled in a single-line flow system via a reservoir. The formed 5-Br-PAPS-Zn(II) complex was measured spectrophotometrically at 552 nm, and the signal intensity corresponded to the zinc concentration. After passing through a flow-through cell, the carrier stream then returned to the reservoir, and the main reagent, 5-Br-PAPS, was successfully regenerated by a ligand-exchange reaction with EDTA, allowing the repetitive determination of zinc. The calibration curve for zinc was linear in the concentration range from 0.4 to 10.0 mg dm(-3) with a correlation coefficient of 0.9995 (n = 6). The detection limit of this method was 0.02 mg dm(-3) (S/N= 3). This method allowed as many as 300 repetitive determinations of 2.0 mg dm(-3) zinc solution with only 100 cm3 of the circulating carrier solution, providing a reduction in the consumption of reagents and an elimination of waste, an important approach towards clean chemistry.
Journal Article↗
The demographic cycle and optimal schooling choices.
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Americas↗
The analysis and assay of cyclic nucleotide phosphodiesterase isoenzyme activity.
Greater knowledge over the past decade on the biochemical properties, as well as the identification of specific pharmacological tools has led to a marked improvement in the methods employed for the analysis and assay of PDE isoenzymes. A major message has been the marked species and tissue-dependent variation in the distribution of the various isoenzymes and their subtypes. This has great implications not only in terms of extrapolating animal data to the human situation, but also from one tissue to another. Thus, it is critical, in particular for drug discovery efforts, to characterize human PDEs in the relevant tissue. Molecular cloning is probably the best and most direct route to achieve this objective since access to disease-free human tissue is heavily limited. Alternatively, well-characterized animal tissues showing PDE and pharmacological profiles similar to human tissues may be utilized. The methods described in this chapter have been successfully applied to study to the biochemistry and pharmacology of PDEs in both animal and human tissues, and in the discovery of novel selective inhibitors for these proteins.
3',5'-Cyclic-AMP Phosphodiesterases↗
3':5'-cyclic adenosine monophosphate phosphodiesterase: negative cooperativity.
Kinetic and chromatographic analysis of cyclic nucleotide phosphodiesterase (EC 3.1.4.c) obtained from rat tissue has revealed that this enzyme exists in at least two molecular forms. After chromatographic separation, one form (cyclic AMP phosphodiesterase) still exhibits kinetics suggestive of the action of either two enzymes or one enzyme under negative cooperative regulation. Computer model studies were undertaken to distinguish between these two possibilities. The matrix method was used to generate the partition functions for (a) the sum of two independent enzymes and (b) one enzyme exhibiting negative cooperative kinetics. The experimental data were fitted to the theoretical models by a nonlinear least-squares computer program. The results show that, while both models can fit the data, the two-enzyme model would require contamination far in excess of what is detectable physically or by activity measurements. Thus, the negative cooperative model seems the more appropriate theoretical explanation of the observed kinetic behavior of this enzyme. The implication of negative cooperativity with respect to the regulation of cyclic AMP concentrations in physiological systems is discussed.
Adipose Tissue↗
[The primary study on the detection of sterigmatocystin by biologic enzyme electrode modified with the multiwall carbon nanotubes].
Sterigmatocystin (ST), the secondary metabolite of many kinds of filamentous fungi, is a potent carcinogen structurally related to the aflatoxins (AFT). With similar chemical structure, sterigmatocystion behaves much the homogeneous properties to aflatoxins, both of these mycotoxins exhibit similar biological properties due to their bisfuranoid structure. Since the common, and even heavier pollution, found in foods and feeds-stuff, sterigmatocystion is more harmful than aflatoxins. The reported detection methods of sterigmatocystion included the Thin-layer Chromatography, the High-Performance-Liquid Chromatography, the Enzyme-Linked Immunosorbant Assay and the PCR detection to the toxic gene, however studies about both easy and inexpensive electro-chemical methods have not been found. Our previous studies had discovered that Sterigmatocystin (ST) exist similar sensitivity towards aflatoxin-detoxifizyme (ADTZ), which we had isolated from a fungus, as aflatoxin does. In this work, the preliminary study on electrochemical analysis and determination of ST with triplet electrode enzyme-biosensor system (Ag/AgCl as the reference electrode, Pt and Au as the pair and work electrode, respectively) was carried out. Multiwall-carbon-nanotube (MWNT) had been used to increase the electron transportation on electrode. In the research, the Au electrode was modified by MWNT-immobilized ADTZ, and then the voltammertric behavior of ST was studied by means of cyclic voltammogram analysis and different pulse analysis. Autoprobe CP Research Atomic Force Microscope and TECNAI 10 Transmission Electron Microscope, had been used to detect the MWNT as well as the surface of MWNT-modified ADTZ. The voltammertric behavior of ST was studied by means of cyclic voltammogram analysis and different pulse analysis. The results show that the red-ox peak potential of ST is at the point of -600 mV, the linear detection range is from 8.32 x 10(-5) to 66.56 x 10(-5) mg/mL, the detection limit is at 8.32 x 10(-5) mg/mL, and the response time is 10 seconds. This study provided a good basic work for further research.
Biosensing Techniques↗
Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis.
Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.
3',5'-Cyclic-AMP Phosphodiesterases↗
Multidrug comparison (lorazepam, triazolam, zolpidem, and zopiclone) in situational insomnia: polysomnographic analysis by means of the cyclic alternating pattern.
Since homogeneous samples of insomniacs are difficult to recruit for pharmacotherapy studies, normal sleepers can be used to assess the protective effect of hypnotic drugs, under standardized nonconducive conditions. In particular, a noisy environment is a typical cause of situational insomnia that can be counteracted by a sedative-hypnotic agent. Six healthy middle-aged subjects (three men and three women), with no complaints about sleep, underwent a completely randomized double-blind series of 10 nocturnal polysomnograms with at least 72-h washout intervals. All subjects received a single dose of placebo, zolpidem 10 mg, zopiclone 7.5 mg, lorazepam 1 mg, and triazolam 0.25 mg both under basal and under perturbed conditions. For each individual, five recordings were carried out under basal conditions (sound pressure level not higher than 30 dB) and five recordings under acoustically perturbed conditions (continuous white noise at 55 dB). Sleep quality was assessed by means of a visual analogue scale (VAS). All recordings were scored according to conventional rules (macro-structure) and cyclic alternating pattern (CAP) methodology (microstructure). Statistical analysis was based on a repeated measures analysis-of-variance design integrated by Bonferroni adjusted probabilities. Under placebo, situational insomnia was confirmed by the significant increase in sleep fragmentation (intrasleep wakefulness) and by the significant enhancement of arousal instability (CAP parameters). In contrast to macrostructural information, CAP parameters were highly sensitive in detecting the perturbing effects of noise (mean CAP rate under placebo, 57%) and the protective action of hypnotic drugs during perturbation (mean CAP rate under active medication, 41%). Microstructural analysis enabled us to discriminate hypnotic drugs from placebo, nonbenzodiazepine compounds from benzodiazepine agents, and zopiclone from zolpidem. The latter, in fact, induced the lowest values of CAP rate both under basal (30%) and under noisy (39%) conditions and determined a significant decrease in electroencephalogram arousals. All CAP parameters were significantly correlated with the visual-analogue-scale scores for sleep quality. The use of CAP methodology in a highly standardized model of situational insomnia can be a valid alternative to conventional sleep scoring for the investigation of drug effects on disturbed sleep.
Adult↗
Conformational studies on cyclic dipeptides.
An analysis of 11 crystal structures of cyclic dipeptides so far reported in the literature is made, with main reference to the internal parameters of these molecules. Preferred conformations of the side chains of cyclic dipeptides with different alpha-amino acid residues have been studied by classical energy calculations. The possible conformations of the DKP ring are also studied. The significance of the non-bonded interaction in deciding the pathway for conformational change has also been investigated. The agreement between theoretical results and experimental observations is quite good, both with respect to the conformation of these molecules as well as the enthalpy difference as estimated from n.m.r. studies between different conformers.
Amino Acid Sequence↗
Receptor tyrosine phosphatase psi is required for Delta/Notch signalling and cyclic gene expression in the presomitic mesoderm.
Segmentation in vertebrate embryos is controlled by a biochemical oscillator ('segmentation clock') intrinsic to the cells in the unsegmented presomitic mesoderm, and is manifested in cyclic transcription of genes involved in establishing somite polarity and boundaries. We show that the receptor protein tyrosine phosphatase psi (RPTPpsi) gene is essential for normal functioning of the somitogenesis clock in zebrafish. We show that reduction of RPTPpsi activity using morpholino antisense oligonucleotides results in severe disruption of the segmental pattern of the embryo, and loss of cyclic gene expression in the presomitic mesoderm. Analysis of cyclic genes in RPTPpsi morphant embryos indicates an important requirement for RPTPpsi in the control of the somitogenesis clock upstream of or in parallel with Delta/Notch signalling. Impairing RPTPpsi activity also interferes with convergent extension during gastrulation. We discuss this dual requirement for RPTPpsi in terms of potential functions in Notch and Wnt signalling.
Animals↗
Comparison between using spectral analysis of electrogoniometer data and observational analysis to quantify repetitive motion and ergonomic changes in cyclical industrial work.
Spectral analysis of continuously measured joint angles using an electrogoniometer was considered as a potentially efficient method for quantifying exposure to physical stress in repetitive manual work. The method was previously demonstrated in the laboratory but has not yet been tested extensively in the field. Spectral analysis was compared against observational analysis, consisting of time-and-motion study and posture classification. Six industrial jobs were selected: (1) press operation, (2) large parts hanging, (3) product packaging, (4) small parts hanging, (5) parts counting and sorting and (6) construction vehicle operation. The posture angle data were synchronized with activities on the video using an interactive multimedia video data acquisition system. Motion for every joint was analyzed using both spectral analysis and observational analysis. Joint angles for the wrist, elbow and shoulder were directly measured using electrogoniometers. Visual posture classification involved determining joint angles from a frozen videotape image sampled three times per s. Repetitiveness was quantified for observational analysis using time study to measure the frequency that specific motions repeat, while spectral analysis measured repetitiveness as the frequency where spectral peaks occurred. Spectral analysis agreed closely with observational analysis. Correlation between the repetition frequencies obtained using time study and spectral analysis was 0.97, with no statistically significant difference observed. Average sustained posture was quantified as the mean, and posture deviation as the RMS angle of joint motion. No statistically significant differences between data obtained using posture classification or spectral analysis were observed for either posture deviation or sustained posture. Since posture classification was very limited in resolution and often contained measurement errors caused by poor joint visibility, the correlation between the postural classification and spectral analysis was 0.77 for sustained posture and 0.53 for posture deviation. When considering only large motions that exceeded the posture classification angle precision, the correlation between postural classification and spectral analysis was 0.81 for sustained posture and 0.81 for posture deviation. Spectral analysis of electrogoniometer data were, therefore, an efficient method for analyzing repetitive manual work that obtained equivalent results, and was more precise than observational analysis.
Electrophysiology↗
Fourier phase analysis in non-cyclic dynamic studies.
Single harmonic Fourier amplitude and phase (FA/P) analyses of 129 radionuclide angiographic (RNA) studies were found to reflect accurately the sequence and quantity of perfusion determined in the independently interpreted RNA studies. In addition, the anatomic detailing in peripheral studies (hands, feet, etc.) was considered superior to that obtained in RNA studies.
Adult↗
Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli.
The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli. Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences. In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product. When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI). Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved. Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations. Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand.
Adenosine Triphosphatases↗
Analysis of a novel cyclic Amp inducible prespore gene in Dictyostelium discoideum: evidence for different patterns of cAMP regulation.
The D7 cDNA clone hybridizes to a 2.8 kb mRNA which first appears at the mound stage of development in the cellular slime mold Dictyostelium discoideum. This gene which is cyclic AMP (cAMP) inducible and is expressed specifically in the prespore cells contains an open reading frame interrupted by only one intron. The predicted amino acid sequence indicates a novel prespore protein which differs from all of the previously described prespore proteins in that it contains no internal repeats and does not share any homology with any of the other prespore genes. The amino acid sequence predicts a protein of 850 amino acids with a molecular weight of 95,343 daltons and an isoelectric point of 4.25. The protein is very rich in glutamine (13.8%), asparagine (10.6%) and glutamic acid (10.4%) with one potential glycosylation site and 28 possible sites for phosphorylation. The amino terminus is hydrophobic with characteristics of a signal sequence while the entire carboxyl half of the protein is notable for its hydrophilicity. Comparison of cAMP regulation of the D7 gene with the regulation of two other cAMP regulated prespore genes, the PL3(SP87) gene and the Psa(D19), reveals some striking differences. Disaggregation in the presence of cAMP results in transient degradation of mRNA for all three genes. The transcription rate for the D7 and PsA(D19) genes remains relatively unaffected by disaggregation but there is a rapid although transient decline in the transcription rate of the PL3(SP87) gene. Although the accumulation of all three mRNAs is first detectable at mound stage, transcription of the D7 and PsA(D19) genes is detected earlier in development, at rippling aggregate stage several hours prior to the earliest time when transcription of the PL3(SP87) gene is detected. Analysis of the promoter region of the D7 gene reveals three CA like boxes flanked by direct repeats as well as four G rich regions that may serve as regulatory elements.
Amino Acid Sequence↗
Automatic detection of cyclic alternating pattern (CAP) sequences in sleep: preliminary results.
OBJECTIVES: The analysis of cyclic alternating pattern (CAP) provides important microstructural information on arousal instability and on EEG synchrony modulation in the sleep process. This work presents a methodology for automatic classification of the micro-organization of human sleep EEG, using the CAP paradigm. METHODS: The classification system is composed of 3 parts: feature extraction, detection and classification. The feature extraction part is an EEG generation model-based maximum likelihood estimator. The detector part for the CAP phases A and B is done by a variable length template matched filter, while the classification criteria part is implemented on a state machine ruled-based decision system. RESULTS AND CONCLUSIONS: The preliminary results of the automatic classifier on a group of 4 middle-aged adults are presented. The high agreement between the detector and visual scoring is very promising in the achievement of a fully automated scoring system, although a more exhaustive evaluation program is needed.
Adult↗
Stereochemical studies on cyclic peptides. Part XI. Conformation of cyclic pentapeptides having intramolecular 3 leads to 1 hydrogen bonds.
Conformational analysis of cyclic pentapeptides having two intra-ring 3 leads to 1 hydrogen bonds has been carried out. It is found that the structure can easily be formed with trans planar peptide units without causing significant angular strain at the alpha-carbon atoms. Four different types of conformations designated Types I--IV are possible for the backbone structure. Details of these four types of conformations and also the accommodating possibility of these types for allglycyl and all-alanyl residues are presented. Three of the four types have relatively low energies for glycyl residues whereas the other one has a slightly higher energy. When alanyl residues are introduced at the five alpha-carbon atoms, the types that are energetically favourable depend upon the sequence of isomers. Energy calculations have also been carried out for the combinations of glycyl, L- and D-alanyl residues. The theoretical results are compared with available experimental observations both from solution and solid state studies.
Amino Acid Sequence↗
Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics.
We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.
Amino Acid Sequence↗