Evidence from its temperature dependence that the β-dielectric dispersion of cell suspensions is not due solely to the charging of a static membrane capacitance

1. A survey of the literature indicates that the apparent capacitance per unit area (C _m ) of biological membranes is in general significantly greater than is that of artificial phospholipid (black lipid) membranes (BLM). It is not possible, by quantitative arguments alone, to exclude that this simply reflects the idea that protein-containing biological membranes have a greater thickness than BLM. 2. The temperature-dependence of the membrane capacitance for both solvent-containing and solvent-free BLM is negative. However, where appropriate data are available, it appears that the capacitance of biological membranes has a positive temperature-dependence, indicating a qualitative difference between natural and artificial membranes. 3. Using a 4-terminal dielectric spectrometer, and the fitting program and electrode polarisation correction described in the accompanying paper, we have carried out a careful study of the temperature-dependence of the -dielectric dispersion of a unicellular eukaryote (Saccharomyces cerevisiae) and a prokaryote (Escherichia coli). 4. In the range 15–40 °C, the temperature-dependence of the -dielectric dispersion (and thus in principle of C _m ) in S. cerevisiae and E. coli is respectively +0.13 and +0.35% (°C)^–1. 5. Flow cytometric measurements indicate that the cell size of E. coli is unchanged in the temperature range studied. 6. These data strongly suggest that the -dielectric dispersion in cell suspensions is not due solely to the charging of a static membrane capacitance. It is proposed that the positive temperature coefficient of the -dispersion reflects the contribution of temperature-dependent, partially restricted, lateral motions of the charged lipid and protein components of the cytoplasmic membrane. Offprint requests to: D. B. Kell     

Dielectric properties of human blood and erythrocytes at radio frequencies (0.2–10 MHz); dependence on cell volume fraction and medium composition

The dielectric properties of human erythrocytes (red blood cells) suspended in whole blood and in isotonic media at various volume fractions (haematocrits) have been studied in the frequency range 0.2–10 MHz, in which the so-called-dispersion due to the Maxwell-Wagner effect is known to occur. The capacitance and conductance at 25 °C were measured by an instrument interfaced to a computer. The rectangular sample cavity (1 ml volume) contained four pure gold electrode pins, and the sample could be circulated by a roller pump. The frequency-dependence of the permittivity and conductivity were fitted by non-linear least squares regression. Corrections were applied for non-linearity in the dielectric increment at high haematocrit, and for electrode polarisation when diluting the blood in saline. Data were interpreted in terms of a simple equivalent resistor-capacitor circuit. From the measured haematological values the specific membrane capacitance (C_m) and the conductivities internal and external to the cells (_i and _o respectively) were estimated. The conductivities behaved in a predictable manner with a mean of 0.458 S · m^–1 (s.d. ± 0.044) for _i, whereas the value of C_m (and indeed the actual capacitance of the suspension) was dependent on the amount of plasma present. Hence, in stationary normal (anticoagulated) whole blood samples, C_m was as high as 2.98 F · cm^–2 (s.d. ± 0.40), in contrast to about 0.9 F · cm^–2 in blood diluted more than two-fold (to less than 20% hct) in isotonic media. The high value remained when the diluent was plasma. The C_m value returned to a high value when washed erythrocytes were reconstituted with plasma, provided that this was present at above a critical or threshold concentration of about 30 vol % in the medium, irrespective of the haematocrit in the range studied (15–44%). The C_m remained low in serum. When added to washed cells in saline, purified fibrinogen had no effect. However, high C_m values were obtained by fibrinogen supplementation to serum and diluted plasma. Applying moderate flow to whole blood approximately halved its high C_m value in an exponential manner with flow rate, whilst the C_m of washed cells (31–67% hct) slightly increased, and converged to the value for whole blood under flow. We interpret the highapparent C_m value in stationary samples to be a result of rapid cell aggregation in the presence of plasma, where rouleaux formation takes place before visible sedimentation sets in.     

Dielectric properties of yeast cells

Summary Dielectric measurements were made on suspensions of intact yeast cells over a frequency range of 10 kHz to 100 MHz. The suspensions showed typical dielectric dispersions, which are considered to be caused by the presence of cytoplasmic membranes with sufficiently low conductivity. Since the conductivity of the cell wall was found to be of nearly the same value as that of the suspending medium, composed of KCl solutions in a range from 10 to 80mm, the cell wall may be ignored in establishing an electrical model of the cells suspended in such media. An analysis of the dielectric data was carried out by use of Pauly and Schwan's theory. The membrane capacitance was estimated to be 1.1±0.1 F/cm², which is compared with values reported so far for most biological membranes. The conductivity of the cell interior was almost unchanged with varying KCl concentrations and showed low values owing to the presence of less conducting particles, presumably intracellular organelles. The relatively low dielectric constant of about 50 obtained for the cell interior, in comparison with values of aqueous solutions, may be attributed also to the presence of intracellular organelles and proteins.     

Microwave Resonant Sensor for Non-invasive Characterization of Biological Tissues

Context

Microwave sensing appears to be an open wide field to investigate medical applications, such as monitoring of vital signs (temperature, arterial pressure, …), following different kinds of pathologies (cancer, glucose level …) and aid for medical diagnosis. It offers an alternative to determine the dielectric properties of biological tissues through the development of local non-invasive and/or embedded sensors, giving thus a kind of imaging by the dielectric contrast. Moreover, RF communications links between several sensors can be developed to realize “Body Area Networks”.

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Tetrachloroethene metabolism of Dehalospirillum multivorans

Dehalospirillum multivorans is a strictly anaerobic bacterium that is able to dechlorinate tetrachloroethene (perchloroethylene; PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) as part of its energy metabolism. The present communication describes some features of the dechlorination reaction in growing cultures, cell suspensions, and cell extracts of D. multivorans. Cell suspensions catalyzed the reductive dechlorination of PCE with pyruvate as electron donor at specific rates of up to 150 nmol (chloride released) min^-1 (mg cell protein)^-1 (300 M PCE initially, pH 7.5, 25°C). The rate of dechlorination depended on the PCE concentration; concentrations higher than 300 M inhibited dehalogenation. The temperature optimum was between 25 and 30°C; the pH optimum at about 7.5. Dehalogenation was sensitive to potential alternative electron acceptors such as fumarate or sulfur; nitrate or sulfate had no significant effect on PCE reduction. Propyl iodide (50 M) almost completely inhibited the dehalogenation of PCE in cell suspensions. Cell extracts mediated the dehalogenation of PCE and of TCE with reduced methyl viologen as the electron donor at specific rates of up to 0.5 mol (chloride released) min^-1 (mg protein).^-1 An abiotic reductive dehalogenation could be excluded since cell extracts heated for 10 min at 95°C were inactive. The PCE dehalogenase was recovered in the soluble cell fraction after ultracentrifugation. The enzyme was not inactivated by oxygen.Abbreviations PCE Perchloroethylene or tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon - MV Methyl viologen     

The “biological ego”. From Garrod's “chemical individuality” to Burnet's “self”

Starting from the conceptual premises of Garrod, who as long ago as 1902 spoke of chemical individuality, and of Burnet (1949), who recognized as self one's own molecular antigenic structures (as opposed to the antigenic alien: the non- self), the discovery and understanding of HLA antigens and of their extraordinarily individual and differentiated polymorphisms have gained universal recognition. Transplant medicine has now dramatically stressed, within man's knowledge of himself, the characteristic of his biological uniqueness. Today man, having become aware of being a biological antigenic-molecular individuality which is unique and different from that of all of his fellow men (except for monozygotic twins), can therefore easily consider himself a true biological Ego.Abbreviations BMT bone marrow transplantation - GVHD graft versus host disease - HLA human leukocyte antigens - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MLR mixed lymphocyte reaction     

Glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1. NeuAc-coupled IL-1 (NeuAc-IL-1) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1 exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1 to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1 was high compared to IL-1. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.     

Inheritance of resistance to blackmold (Alternaria alternata (Fr.) Keissler) in two interspecific crosses of tomato (Lycopersicon esculentum ×L. cheesmanii f.typicum)

Inheritance of resistance to blackmold, a disease of ripe tomato fruit caused byAlternaria alternata, was studied in two interspecific crosses. The parents, F₁ and F₂ generations of a cross between the susceptibleLycopersicon esculentum Mill. cultivar Hunt 100 and the resistantL. Cheesmanii f.typicum Riley accession LA 422, and the parents, F₁, F₂, F₃, and BC₁ P₂ generations of a cross between the susceptibleL. Esculentum cv. VF 145B-7879 and LA 422 were evaluated. The following disease evaluation traits were used: symptom rating (a symptom severity rating based on visual evaluation of lesions), diseased fruit (the number of diseased fruits divided by the total number of fruit scored), and lesion size (a function derived from the actual lesion diameter). Generation means analysis was used to determine gene action. The data of the Hunt 100 × LA 422 cross fit an additive-dominance model for all three traits. The VF 145B-7879 × LA 422 cross data best fit a model that included the additive × additive and additive × dominance interaction components for the trait diseased fruit, whereas higher-order epistatic models would have to be invoked to fit the data for the traits symptom rating and lesion size. A minimum of one gene segregated for all three traits. Broad-sense heritability estimates ranged from 0.09 to 0.16 for all three traits, indicating that selection for improved resistance to blackmold will require selection on a family performance basis.     

Dielectric characterization of costal cartilage chondrocytes

Background

Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

Methods

Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes.

Results

The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance.

Conclusions

We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues.

General significance

The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.     

Hypoxia Tolerance of Two Haplochromine Cichlids: Swamp Leakage and Potential for Interlacustrine Dispersal

The ability to tolerate hypoxia in some haplochromine cichlid fishes contributes to the richness of habitats occupied by the lineage and may be important in interlacustrine dispersal through swampy channels. Lacustrine members of the genus Astatotilapia tend to be ecologically plastic but are rarely encountered in the interior of dense swamps. A notable exception is seen in the swamp corridor that joins Lake Kabaleka with Lake George, Uganda, where one species (Astatotilapia wrought-iron) is abundant, and a second species, A. aeneocolor, is rare. Both species are abundant in the open waters of the main lake. In this paper, we compare physiological (oxygen consumption) and behavioral indicators of hypoxia tolerance between A. wrought-iron from swamp and open-water habitats and between the two species of Astatotilapia. When exposed to progressive hypoxia, all fish used aquatic surface respiration (ASR); however, swamp-dwelling A. wrought-iron showed lower gill ventilation rates prior to the initiation of ASR, higher pre-ASR aggression rates, higher swimming speed during ASR, and a higher rate of bubble exchange than both the open-water group of A. wrought-iron and A. aeneocolor. These differences may reflect interpopulational variation in selection pressure for low-oxygen tolerance between swamp and open-water habitats. Several lines of evidence suggest that A. wrought-iron was in general more hypoxia tolerant than A. aeneocolor. These include a lower ASR₉₀ threshold, a drop in gill ventilation rate with the onset of ASR, and lower rate of equilibrium loss under extreme hypoxia in A. wrought-iron. The routine metabolic rate and critical oxygen tension did not differ between swamp-dwelling and open-water A. wrought-iron, or between A. wrought-iron and A. aeneocolor. Comparative data on the ASR thresholds and critical oxygen tensions of the Astatotilapia species from Lake Kabaleka and other East African cichlids suggest intermediate hypoxia tolerance. Nevertheless, our study suggests that some generalized lacustrine haplochromines may leak through swamp corridors even under relatively extreme conditions.     

A posteriori time-varying filtering of averaged evoked potentials

The problem of estimating an unknown transient signal, given an ensemble of waveforms, in which this signal appears as a nonrandom component in the presence of additive noise is considered. This problem is solved by generalizing the method of a posteriori Wiener filtering. In the new method, the ensemble average is filtered by a time-varying system which is based on estimated time-varying power spectra of signal and noise. The nature of this system, and the computational procedures involved, are discussed in detail. A software package for time-varying filtering is briefly described. Application of the method is illustrated by a simulation example, which also provides a comparison to time-invariant a posteriori Wiener filtering.     

Fine structure of Methanomonas methanooxidans

Résumé Chez 5 bactéries anaérobies facultatives: Providencia alcalifaciens, Edwardsiella tarda, Aeromonas hydrophila, mutants catégorie 1 de Providencia stuartii et Hafnia, la nitrate-réductase B a un caractère inductible et sa biosynthèse est réprimée par O₂. Chez Micrococcus denitrificans, elle est constitutive et non répressible par NH₄ ⁺ ou par O₂. Dans le cas d'une bactérie aérobie stricte qui assimile le nitrate: Pseudomonas putida, elle est constitutive (ou faiblement inductible), non répressible par NH₄ ⁺, atteint un niveau plus élevé dans les cultures sur milieu complexe que dans les cultures sur milieu synthétique.O₂ exerce une action sur le fonctionnement de la nitrate-réductase B: l'aération inhibe réversiblement et de façon presque complète la réduction de NO₃ ^- en NO₂ ^-, aux dépens du glucose comme donneur d'électrons, par des suspensions cellulaires d'E. tarda, M. denitrificans, P. putida, et du mutant de Hafnia.

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Bacterial nitrate reductasesIV. Regulation of the biosynthesis and activity of enzyme B

Summary 5 facultative anaerobic bacteria (Providencia alcalifaciens, Edwardsiella tarda, Aeromonas hydrophila, and category 1 mutants of Providencia stuartii and Hafnia) form nitrate reductase B inducibly; the biosynthesis of the enzyme is repressed by O₂. In Micrococcus denitrificans, the enzyme is constitutive and its intracellular level is unaffected by the presence either of NH₄ ⁺ or O₂. A strict aerobe that assimilates nitrate, Pseudomonas putida, similarly forms nitrate reductase B constitutively, although the level of the enzyme is higher in cultures grown on complex than on synthetic media.O₂ affects the activity of nitrate reductase B: the reduction of NO₃ ^- to NO₂ ^-, with glucose as electron donor, by suspensions of E. tarda, M. denitrificans, P. putida and the mutant of Hafnia, is almost totally but reversibly inhibited by aeration.

     

Recycling of spent shiitake substrate for production of the oyster mushroom,Pleurotus sajor-caju

Pleurotus sajor-caju was produced on a basal medium containing spent shiitake substrate plus 10% wheat bran and 10% millet. An analysis of the fibrous composition of the spent shiitake substrate revealed that 85% of the original hemicellulose, 44% of the original cellulose and 77% of the original lignin was not consumed during production of a full crop (78% biological efficiency) of shiitake (63-day harvest period). To produce P. sajor-caju, the spent shiitake substrate was ground, air dried, supplemented, pasteurized with live steam and spawned. Highest yields (79% biological efficiency) of P. sajor-caju were obtained by supplementing the spent shiitake basal medium with 12% soybean and 1% CaCO₃. Increases in percentage biological efficiency and mushroom size were positively correlated with increasing levels of CaCO₃ added to the basal medium.     

Functional mitochondria in snake Bothrops alternatus erythrocytes and modulation of HbO2 affinity by mitochondrial ATP

Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca²⁺ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca²⁺ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O₂ affinity, which suggests that ATP production by mitochondria may participate in modulation of O₂ transport by hemoglobin.Abbreviations membrane potential - BAE Bothrops alternatus erythrocytes - DNP 2,4-dinitrophenol - DPG 2,3-diphosphoglycerate - EGTA ethyleneglycol tetra-acetic acid - FCCP carbonylcyanide p-trifloromethoxyphenylhydrazone - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TPP⁺ tetraphenylphosphonium - TRIS tris-(hydroxymethyl)aminomethane     

A rapid microbead method for breaking pathogenic mycobacteria: Application in SDS-PAGE and western blot analysis

A rapid method used to obtain cell-free extracts from pathogenic mycobacteria was successfully applied to five slowly growing species. The method, which simply consisted of vortexing of the bacterial suspensions with 130-m glass microbeads under predefined conditions, resulted in the breaking of bacterial cells as evidenced by electron microscopy and gave about 1 mg/ml of total proteins. The cell-free extracts so obtained were suitable for SDS-PAGE analysis as well as Western blots. In a pilot test system, this method was applied toMycobacterium avium (M. av),M. paratuberculosis (P.tb), and related species isolated from wood-pigeon (M. silvaticum) and Crohn's disease (CD). The procedure was used in the analysis of various mycobacterial proteins by SDS-PAGE and Western blotting, and the findings were compatible with recently published data using criteria based on DNA polymorphism and numerical taxonomy.     

Rhythmicity of swimming activity in an axenic population of Paramecium multimicronucleatum

A unicellular organism, Paramecium, exhibits circadian rhythm activities in many physiological phenomena, i.e., mating reactivity, photoaccumulation in Paramecium bursaria and mating type reversals in Paramecium multimicronucleatum. In this study, we used an image-processing system to analyze swimming activity in a population of Paramecium multimicronucleatum cultured axenically under 12 h-light/12 h-dark cycles (LD 1212). Swimming behavior was recorded both under LD 1212 and constant darkness and images tracing the tracks of Paramecium were produced every 4 min. Swimming activity was represented by the occupied area by the tracks relative to the total observed area. It is high during daytime and low at night and exhibits a freerunning rhythm in constant darkness. Furthermore, criteria for two major components of swimming behavior, straight and circle swimming, were established and analyzed. The results indicate that swimming behavior alters depending on the time of day: straight swimming increased during the day and circling was dominant around dusk both under LD 1212 and constant darkness.Abbreviations ZT Zeitgeber Time - LD 1212 12h- light/12h-dark cycles - TF transversing frequency     

Investigation of the membrane lesion induced in vitro by two mosquitocidal δ-endotoxins ofBacillus thuringiensis

Bacillus thuringiensis produces insecticidal crystalline -endotoxins during sporulation. Leakage of radiolabeled markers from insect tissue culture cells and erythrocytes supports a mechanism of colloid osmotic lysis for the 23K and 27K polypeptides from the -endotoxins ofBacillus thuringiensis var.darmstadiensis 73-E10-2 and var.israelensis, respectively. Both toxins produce a plasma membrane pore 0.6–1.0 nm in radius.     

Relation of nitrogen fertilizers and manure to vascular wilt of egyptian cotton caused by four fusaria species and strains

Summary Four different species and strains ofFusarium, namely,F. oxysporum f.vasinfectum (Atk.)Snyder &Hansen,F. solani (Mart.)App. &Wr.,F. vasinfectum (Atk.) f₁ Wr. strainFahmy, andF. vasinfectum Atk. are responsible for cotton wilt in U.A.R. Pathogenicity experiments have shown that the four experimentalFusaria possess varying degrees of pathogenicity towards two susceptible cotton varieties, namely, Karnak and Bahtim 190. On the other hand, Ashmouni cotton proved resistant to the fourFusaria. Variability of pathogenicity of the experimentalFusaria is not only related to host cotton plant or parasite, but also to nitrogenous fertilizer or manurial treatment of the soil. Resistance of Ashmouni cotton was not broken in presence of sodium nitrate, ammonium nitrate or stable manure added to soil inoculated with any of the fourFusaria. The application of nitrogen fertilizers or manure resulted on the whole, in increased wilt-disease incidence of the susceptible cotton varieties. Stable manure enhanced predisposition to vascular-wilt more than other nitrogen treatments, especially in presence of the virulentF. oxysporum f.vasinfectum or both strains ofF. vasinfectum Atk. Ammonium nitrate induced the sudden appearance of mottling characteristic of vascular-wilt of cotton when Bahtim 190 plants were raised in nitrogen-fertilized soil inoculated withF. solani (Mart.)App. &Wr. At the same time, the raised affected plants showed increased growth-vigour and enhanced maturity of their leaves.     

Activation of RAT erythrocyte phosphofructokinase by AMP and by non-physiological concentrations of Cyclic AMP

Summary Cyclic AMP (300µ m) activates phosphofructokinase from dialyzed haemolysates of mature rat erythrocytes. The main conclusions are: a) Cyclic AMP, at pH 7.1 and low concentrations of fructose-6-phosphate, is able to reverse the inhibition produced by different amounts of ATP (up to 1.5mm). b) The cyclic nucleotide is a positive allosteric effector of the enzyme as shown by the displacement of sigmoidal fructose-6-phosphate saturation curve to hyperbolic kinetics in the presence of inhibitory concentrations (1.5mm) of ATP. c) Cyclic AMP has no significant influence as deinhibitor of phosphofructokinase either at pH 7.1 and non-inhibitory levels (0.25mm) of ATP or at pH 8.1 and inhibitory (1.5mm) of non-inhibitory (0.25mm) concentrations of ATP. Similar conclusions were obtained with 300µ m AMP but not at a lower concentration (3µ m) with both nucleotides.The comparison of cyclic AMP results with those obtained under similar concentrations of AMP suggest that cyclic AMP is really only an in vitro modulator of the enzyme from rat erythrocytes, presumably at an AMP regulatory site, since non-physiological concentrations are required to act as deinhibitor.