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Methodological variables during analysis of in vivo cerebellar cyclic GMP, an indirect marker of nitric oxide release.

In light of the recent recognition of the physiological significance of nitric oxide, there is considerable interest in the methodological variables that can confound the results of the cerebellar cGMP analysis from in vivo experiments. In this study, using male Swiss Webster mice, the effect of such methodological variables as 1) weight of the animals; 2) tissue extraction procedures used in radioimmunoassay for cGMP; and 3) the commercial source of the assay kit on, harmaline-, pentylenetetrazole- or SNAP-induced increase in cerebellar cGMP in vivo were evaluated. Results indicate that mice in the 15- to 19-g weight range are most sensitive and best suited for in vivo drug effects on cerebellar cGMP. Furthermore, for the extraction of cerebellar cGMP, use of ice-cold 0.5N hydrochloric acid and subsequent dilution of the sample in assay buffer is the simplest and fastest method. Present data also indicate that the source of the radioimmunoassay kit has a significant effect on the cerebellar cGMP results. Based on the present results, the protocol developed and the guidelines drawn are timely and of high practical significance for research in the area of pharmacology of nitric oxide.

Animals↗

Correlation of granulocyte intracellular activities of cyclic nucleotide phosphodiesterases with leukocyte count in patients with chronic myelogenous leukaemia.

The activity of adenosine cyclic 3':5'-monophosphate phosphodiesterase in granulocytes of patients with CML essentially depends on the granulocyte donor's WBC count. The ratio of cAMP-PDE/cGMP-PDE activities in CML granulocytes strongly correlates with CML host WBC count. The regression analysis of cyclic nucleotide phosphodiesterase activities and counts of individual constituents of the white blood cell population present in the blood of CML patients showed the primary relationship between the natural logarithm of total WBC count and the cAMP-PDE/cGMP-PDE activity. The results suggest that the properties of CML granulocytes depend on the accumulation of these cells in the CML host.

3',5'-Cyclic-AMP Phosphodiesterases↗

Human endometrial proteins with cyclic changes in the expression during the normal menstrual cycle: characterization by protein sequence analysis.

Endometrial proteins showing cyclic expression during the normal menstrual cycle were localized on two-dimensional (2-D) electrophoresis gels separating proteins with isoelectric points (pl) ranging from 3.5 to 7 and relative molecular weights ranging from 10 to 300 kDa. Menstrual cycle-related proteins were excised from several 2-D gels, concentrated by one-dimensional (1-D) sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and cleaved in situ by trypsin. The tryptic fragments were extracted and separated by reverse phase high performance liquid chromatography (RP-HPLC). Finally, the partial amino-terminal amino acid sequence of selected tryptic fragments were determined for each protein. We aimed at characterizing the 21 menstrual cycle-related proteins that were visible on silver-stained 2-D electrophoresis gels. Of the proteins being maximally synthesized in the proliferative phase endometrium, we identified proteins associated mainly with the cytoskeleton: vimentins, keratin, tropomyosin and tubulin, but also proteins such as proliferating cell nuclear antigen and beta-galactoside binding lectin. The partial amino acid sequences for another two proteins did not match any protein sequence in the Protein Identification Resource (PIR) and Swissprot databases. In the group of proteins having maximal synthesis in the secretory phase endometrium, we identified creatine kinase chain B and an isocitrate dehydrogenase-homologous protein, both of which are involved in energy metabolism. However, we also identified the annexin IV precursor, the 14-3-3 protein homologue also called stratifin or the epithelial cell marker protein 1 and the 21K tumour protein. Finally, four of the proteins were present in too low amounts to allow characterization. Interestingly, most of the identified proteins have not previously been described as having a menstrual cycle-related synthesis in the human endometrium. It may be considered that the concentration of some of the cycle-related proteins may be used in clinical situations to reflect specific endometrial phases.

Amino Acid Sequence↗

Cyclic dibenzoylhydrazines reproducing the conformation of ecdysone agonists, RH-5849.

We have investigated the biologically active conformation of the non-steroidal ecdysone agonist, 1-tert-butyl-1,2-dibenzoylhydrazine (RH-5849) by means of design, synthesis and conformational analysis of cyclic derivatives of RH-5849. Among the synthesized compounds, a 6-membered cyclic hydrazine bearing two benzoyl groups (5) exists in three conformational states in solution, and the major unsymmetrical conformer of 5 is similar to that of RH-5849 on the basis of 1H NMR and X-ray analyses. The 3,3-dimethyl derivative of 5 (10) exists as a single unsymmetrical conformer. Although there is conformational similarity of the cyclic derivatives with RH-5849, these compounds did not show any hormonal or insecticidal activity. The hydrogen bonding character of the amide N-H group of the dibenzoylhydrazine seems to play a critical role in the appearance of the biological activity.

Animals↗

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.

Animals↗

Capital accumulation, aspiration adjustment, and population growth: limit cycles in an Easterlin-type model.

"One of the recent interesting hypotheses of population growth is due to Easterlin who suggests the possibility of self-generating fluctuations in birth numbers. The present paper tries to answer the question whether feedback mechanisms produce persistent oscillations in population growth. A system of two nonlinear differential equations for the per capita capital stock and the aspiration level is studied by a phase portrait analysis. Using the Poincare-Bendixson theorem we derive sufficient conditions for the existence of a stable limit cycle." (SUMMARY IN FRE)

Demography↗

Migration reversals in perspective: the long-wave evidence.

"Although the pattern of polarization reversal reported by Vining and Pallone in the 1970s and the re-emergence of core-ward net migration that they now report for the 1980s are problematic if viewed from the perspective of a short-term time horizon, they are easily understandable in a long-wave context. Evidence is provided for 55-year waves of urbanward migration [in the United States], each of which reached its nadir during the nation's major stagflation crises. The periodic repetitions of the phenomena described by Vining and Pallone suggest the relevance of the interpretations provided by long-wave theory."

Americas↗

Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens.

Geobacter sulfurreducens is capable of anaerobic respiration with Fe(III) as a terminal electron acceptor via a membrane-bound Fe(III) reductase activity associated with a large molecular mass cytochrome c. This cytochrome was purified by detergent extraction of the membrane fraction, Q-Sepharose ion-exchange chromatography, preparative electrophoresis, and MonoQ ion-exchange chromatography. Spectrophotometric analysis of the purified cytochrome reveals a c-type haem, with no evidence of haem a, haem b or sirohaem. The cytochrome has an M(r) of 89000 as determined by denaturing PAGE, and has an isoelectric point of 5.2 as determined by analytical isoelectric focusing. Dithionite-reduced cytochrome can donate electrons to Fe(III)-nitrilotriacetic acid and synthetic ferrihydrite, thus demonstrating that the cytochrome has redox and thermodynamic properties required for reduction of Fe(III). Analysis using cyclic voltammetry confirmed that the reduced cytochrome can catalytically transfer electrons to ferrihydrite, further demonstrating its ability to be an electron transport mediator in anaerobic Fe(III) respiration. Sequence analysis of a cloned chromosomal DNA fragment revealed a 2307 bp open reading frame (ferA) encoding a 768 amino acid protein corresponding to the 89 kDa cytochrome. The deduced amino acid sequence (FerA) translated from the open reading frame contained 12 putative haem-binding motifs, as well as a hydrophobic N-terminal membrane anchor sequence, a lipid-attachment site and an ATP/GTP-binding site. FerA displayed 20% or less identity with amino acid sequences of other known cytochromes, although it does share some features with characterized polyhaem cytochromes c.

Amino Acid Sequence↗

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells.

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.

Cells, Cultured↗

Localization of cyclic nucleotide phosphodiesterase in the multicellular stages of Dictyostelium discoideum.

Cyclic 3',5'-adenosine monophosphate (cAMP) is secreted as the chemotactic signal by aggregating amoebae of the cellular slime mold Dictyostelium discoideum. We have used ultramicrotechniques in the biochemical analysis of cyclic nucleotide phosphodiesterase (PD) distribution in individual aggregates at various stages of development. With handmade constriction pipettes in microliter volumes, sections of lyophilized individuals weighing 20-100 ng could be assayed in a reaction coupled to 5'-nucleotidase. Phosphodiesterase activity was measured at pH 7.5 with 12 microM cAMP, cAMP-PD activity in aggregates ranged from 20-40 mmol/h/kg. In the pseudoplasmodium it had dropped to 5-10 mmol/h/kg and a difference in activity between the anterior prestalk cells and posterior prespore cells began to appear. The utmost posterior sections showed elevated phosphodiesterase from this stage onward. During culmination, activity rose to 40-60 mmol/h/kg associated with the developing stalk, while it declined in the spore mass. The papilla remained constant at 5-10 mmol/h/kg. The pattern of localization in the stalk was the same when cGMP was used as substrate. Extracellular phosphodiesterase inhibitor produced at the aggregation stage was found to reduce the localized activity in the culmination stage by 50-80%, with the most marked inhibition occurring in the center of the papilla. We found no evidence of endogenous heat-stable phosphodiesterase inhibitor within the culminating sorocarp.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

Comparison of continuous and cyclic pumping from a well.

Two practically applicable pumping patterns for wells are: (1) continuous pumping at a sustainable constant rate; and (2) pumping at a sustainable constant rate for a fixed period every day (cyclic pumping). Theoretical analysis shows that with the cyclic pumping pattern the pump can be run at a higher rate than with continuous pumping. However continuous pumping at the maximum continuously sustainable constant rate produces considerably more water. If the objective is to produce as much water as possible, it is important to use a pump designed for running at the maximum continuously sustainable rate.

Models, Theoretical↗

Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase.

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.

3',5'-Cyclic-AMP Phosphodiesterases↗

Cycles in the two-sex problem: an investigation of a nonlinear demographic model.

"A fundamental shortcoming of classic stable population theory is its failure to handle populations differentiated by sex. The classic theory is linear while the two-sex problem is inherently nonlinear. Previous two-sex investigations have focused on equilibrium conditions rather than dynamics, and ignored competition between age groups for marriage partners. This study makes a start at analyzing dynamics and models that incorporate competition, which can play an important role in any realistic marriage model and can turn a model with a stable equilibrium sex ratio into one with a cycling equilibrium." This is a revised version of a paper originally presented at the 1990 Annual Meeting of the Population Association of America. (SUMMARY IN FRE)

Demography↗

Cyclic AMP-dependent protein kinase content of murine T and B lymphocytes.

Murine splenic lymphocytes have been separated by non-activating procedures into enriched T and B cell populations. Cyclic AMP-dependent protein kinase (cAPK) activity has been measured in those separated cell types by histone phosphorylation. As in other tissues, lymphocyte cAPK activity was found to be predominantly a soluble enzyme. By an analysis of cyclic AMP (cAMP) activation of enzyme activity it was shown that: (1) the basal activities were indistinguishable between these major lymphocyte subpopulations, (2) the Ka values for cAMP activation were identical for the two cell types (24 nM cAMP), and (3) the Vmax for enzyme activity was higher in T (5.47 pmole/min/10(6) cells) than in B (3.58 pmole/min/10(6) cells) cells. DEAE-cellulose chromatography experiments with mixed and enriched splenic lymphocyte populations extended and amplified this quantitative difference between T and B cells.

Animals↗

Site-selective lateral multilayer assembly of bienzyme with polyelectrolyte on ITO electrode based on electric field-induced directly layer-by-layer deposition.

We describe here a new approach to construct a multilayer enzyme/polyelectrolyte film on a structured transparent indium-tin oxide (ITO) covered glass electrode surface as micropattern, on which two different types of enzyme distributed laterally on one common substrate without interference. The multilayer film was prepared by alternate electric field directed layer-by-layer assembly deposition and alternate deposition of different redox enzymes and polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA) onto the site-selective ITO glass electrode surface. The cyclic voltammogram, obtained from the ITO glass electrode modified with the glucose oxidase (GO(X))/PDDA and catalase (CA(T))/PDDA multilayers, revealed that the bioelectrocatalytic response is directly correlated to the number of deposition bilayers. From the analysis of cyclic voltammetric characterization, the coverage of catalytically active enzymes per enzyme/PDDA bilayer during the multilayer formation was homogeneous, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, from the atomic force microscopy and Brewster angle microscopy measurements, more information of the multilayer constructed by different methods on the modified electrode surface is obtained and compared. This fabrication technique is simple and would be applicable to the construction of a thickness- and area-controlled biopattern composed of multi-enzymes as well as multiple biomaterials.

Biosensing Techniques↗

A pooled data analysis on the use of intermittent cyclical etidronate therapy for the prevention and treatment of corticosteroid induced bone loss.

OBJECTIVE: To conduct a pooled data analysis in a group of patients defined by sex, menopausal status, and underlying disease in order to examine the effect of intermittent cyclical etidronate in the prevention and treatment of corticosteroid induced osteoporosis. METHODS: We selected 5 randomized, placebo controlled studies that examined the efficacy of intermittent cyclical etidronate therapy in which the raw data were available for analysis. Three were prevention studies and 2 treatment studies. The primary outcome was the difference between treatment groups in the percentage change from baseline in lumbar spine bone density. Secondary outcomes included the difference between treatment groups in the percentage change from baseline in femoral neck and trochanter bone density, and vertebral fracture rates. RESULTS: Results are separately pooled for the prevention and treatment studies. The prevention studies had significant mean differences (95% CI) between groups in mean percentage change from baseline in lumbar spine, femoral neck, and trochanter bone density of 3.7 (2.6 to 4.7), 1.7 (0.4 to 2.9), and 2.8% (1.3 to 4.2) after one year of treatment, in favor of the etidronate group. The treatment studies displayed a mean difference between groups in mean percentage change from baseline in lumbar spine bone density of 4.8 (2.7 to 6.9) and 5.4% (2.5 to 8.4) after one and 2 years of therapy. In the prevention studies, a reduced fracture incidence was observed in the etidronate group compared with the placebo group (relative risk 0.50; CI 0.21 to 1.19). CONCLUSION: Etidronate therapy was effective in preventing bone loss in the prevention studies and in preventing or slightly increasing bone mass in the treatment studies. A fracture benefit was observed in postmenopausal women treated with etidronate in the prevention studies.

Adult↗