Mg Of Cells Research Articles

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.

Catalytic properties of phenol carboxylase In vitro study of CO2: 4‐hydroxybenzoate isotope exchange reaction

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.

Intracellular free calcium concentration and thromboxane A2formation of vascular smooth muscle cells are influenced by fish oil and n-3 eicosapentaenoic acid

The effect of fish oil and n-3 eicosapentaenoic acid (EPA) on intracellular free calcium concentration ([Ca2+]i) and thromboxane B2 (TXB2) formation in resting and stimulated cultured rat vascular smooth muscle cells (VSMC) was examined. In resting control cells [Ca2+]i was 147 +/- 15 nmol l-1 (mean +/- SEM, n = 4). After pretreatment of the cells with fish oil or EPA for 24 days the resting [Ca2+]i was decreased to 126 +/- 10 nmol l-1 and 84 +/- 8 nmol-1, respectively. After stimulation of untreated control cells with either 100 nmol l-1 angiotensin II (AII), 40 micrograms ml-1 low-density lipoprotein (LDL), or 100 ng ml-1 of recombinant platelet-derived growth factor (PDGFAB), [Ca2+]i was (in nmol l-1) 306 +/- 31, 217 +/- 25 and 213 +/- 16. Treatment of cells with fish oil or EPA reduced the stimulatory effect of the agonists, and the following [Ca2+]i values (in nmol l-1) were found: 199 +/- 21, 131 +/- 10, 148 +/- 13; and 175 +/- 11, 98 +/- 12, and 103 +/- 6, respectively. PDGFAB induced a four fold increase in TXB2-generation (270 +/- 28 pg mg-1 cell protein compared with 61 +/- 8.2 pg mg-1 in unstimulated control cells) within 6 min. In cells pretreated with fish oil or EPA, TXB2-formation was reduced by 54% and 44%, respectively. in rat VSMC stimulated by a variety of vasoactive agonist, fish oil and EPA can markedly attenuate intracellular mechanisms related to changes of cytosolic calcium concentration and eicosanoid production.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple isocratic ion-pair high-performance liquid chromatographic determination of 1-β-D-arabinofuranosylcytosine 5′-triphosphate for intracellular drug-monitoring and in vitro incubation assays

A sensitive isocratic ion-pair HPLC method using a reversed-phase C18 column and phosphate buffer at pH 6 for the determination of the cytotoxic intracellular anabolite 1-beta-D-arabinofuranosylcytosine-triphosphate (Ara-CTP) of the antineoplastic drug cytarabine in leukaemic cells in vivo is described. Simultaneous determination of the physiological nucleotide deoxycytidine-triphosphate is possible. The recovery from cells is greater than 90%, the limit of detection is 25 ng ml-1 (51 nM l-1) and about 10 pM mg-1 cell protein. Extraction procedure and pitfalls are discussed. The method enables intracellular drug monitoring of Ara-CTP with standard HPLC equipment.

Optimal temperature control policy for a two-stage recombinant fermentation process.

The optimal temperature control policy to be followed in the operation of a two-stage fermentation system in which gene expression is induced by a temperature-sensitive gene switching system was studied. A genetically structured model was used to describe product formation, and kinetic equations based on experimental data were used to quantify the specific gene expression rate and parameters that affect plasmid instability. A constant temperature control policy and temperature profiling control policy including temperature cycling were studied and compared. Maximum average production rate was obtained from a temperature control policy in which the second stage was operated initially at about 40.5 degrees C and the temperature decreased slightly to a constant value at 40.0 degrees C. The maximum average production rate, which corresponds to the optimal temperature control policy, for an operation of 180 h was 29.7 units of protein (mg of cells)-1 h-1.

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.

Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes

We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.

Cytochrome P450 in highly purified suspension of nonparenchymal liver cells.

Rat liver nonparenchymal cells (NPC) were prepared by pronase digestion and purified on discontinuous gradients on Nycodenz. Morphological and biochemical characterization of cell suspensions showed that they were free of contamination by hepatocytes. We have confirmed the usefulness of pyruvate kinase activity in monitoring the degree of hepatocyte contamination of NPC and we have derived an equation which allows this carry-over to be calculated. Using highly purified suspensions of NPC we have shown that they contain glucose-6-phosphatase in low but detectable levels. Spectrophotometric studies showed that they contain cytochrome P450, with a specific content of 24 +/- 5 pmole mg-1 cell protein. A potential source of error in previous studies was recognized; namely that peroxidase, present in NPC in high concentration, is able to mask the absorption due to cytochrome P450. Both the presence and inducibility of this enzyme in NPC prepared from rats pretreated with phenobarbital or 3-methylcholanthrene have been confirmed using Western blot analysis.

Enhancement of Inoue-Melnick virus synthesis by 5-bromodeoxyuridine in human meningioma (MG-1) cells.

The authors found remarkable enhancement of Inoue-Melnick virus (IMV) synthesis by 5-bromodeoxyuridine (BUDR) in human meningioma (MG-1) cells, a virus-producer line of type 1 IMV. Treatment with BUDR resulted in rapid and abundant synthesis of infectious IMV in MG-1 cells. The titer of the cell-associated virus in treated cells increased approximately 6.0 log 10 compared with that in untreated cells. Immunofluorescent antibody tests revealed that IMV-associated late antigen was induced by BUDR in the cytoplasm of approximately 50% of treated cells. A clonal difference was also found in the enhancement of BUDR on the IMV synthesis in MG-1 cells. The most remarkable enhancing effect of BUDR was observed in the clone C line, and a herpes-type virus was detected by negative-staining electron microscopy in the culture fluid of the clone C treated with BUDR.

Energy-dependent, high-affinity transport of nickel by the acetogen Clostridium thermoaceticum.

The nickel transport system of Clostridium thermoaceticum was investigated with 63NiCl2 and an anaerobic microfiltration transport assay. Transport was optimal at pH 7 to pH 7.5 and 65 degrees C and decreased in the presence of metabolic uncouplers and inhibitors. Exogenous nickel was concentrated 3,000-fold over the apparent nickel concentration gradient during typical transport assays. Stored cellular energy appeared to provide a short-term energy source to power nickel transport, and starvation experiments demonstrated external energy source stimulation of nickel translocation. The apparent Km and Vmax for nickel transport by carbon monoxide-dependent chemolithotrophic cells approximated 3.2 microM Ni and 400 pmol of Ni transported per min per mg of cells (dry weight), respectively. Magnesium, calcium, cobalt, iron, manganese, and zinc did not inhibit the transport of nickel.

Synthesis of l-Dopa by Escherichia intermedia cells immobilized in a carrageenan gel

Escherichia intermedia cells were immobilized by entrapment in a carrageenan gel and used for l-DOPA synthesis from catechol, pyruvate, and ammonia. A preparation containing 75 mg of cell per gram of gel retained 60–65% of its original activity. The effect of substrate concentrations on the initial rate of l-DOPA synthesis was very similar for free and immobilized cells, and substrate inhibition was observed for the three substrates. In batch reactors, up to 7.8 g l −1 of l-DOPA was obtained in 20 h (productivity 0.39 g l −1 h −1). Cells immobilized in a carrageenan gel showed higher l-DOPA synthesis, in both initial rates conditions and batch reactors, than cells immobilized in a polyacrylamide gel.

Pathway and sites for energy conservation in the metabolism of glucose by Selenomonas ruminantium.

On the basis of enzyme activities detected in extracts of Selenomonas ruminantium HD4 grown in glucose-limited continuous culture, at a slow (0.11 h-1) and a fast (0.52 h-1) dilution rate, a pathway of glucose catabolism to lactate, acetate, succinate, and propionate was constructed. Glucose was catabolized to phosphoenol pyruvate (PEP) via the Emden-Meyerhoff-Parnas pathway. PEP was converted to either pyruvate (via pyruvate kinase) or oxalacetate (via PEP carboxykinase). Pyruvate was reduced to L-lactate via a NAD-dependent lactate dehydrogenase or oxidatively decarboxylated to acetyl coenzyme A (acetyl-CoA) and CO2 by pyruvate:ferredoxin oxidoreductase. Acetyl-CoA was apparently converted in a single enzymatic step to acetate and CoA, with concomitant formation of 1 molecule of ATP; since acetyl-phosphate was not an intermediate, the enzyme catalyzing this reaction was identified as acetate thiokinase. Oxalacetate was converted to succinate via the activities of malate dehydrogenase, fumarase and a membrane-bound fumarate reductase. Succinate was then excreted or decarboxylated to propionate via a membrane-bound methylmalonyl-CoA decarboxylase. Pyruvate kinase was inhibited by Pi and activated by fructose 1,6-bisphosphate. PEP carboxykinase activity was found to be 0.054 mumol min-1 mg of protein-1 at a dilution rate of 0.11 h-1 but could not be detected in extracts of cells grown at a dilution rate of 0.52 h-1. Several potential sites for energy conservation exist in S. ruminantium HD4, including pyruvate kinase, acetate thiokinase, PEP carboxykinase, fumarate reductase, and methylmalonyl-CoA decarboxylase. Possession of these five sites for energy conservation may explain the high yields reported here (56 to 78 mg of cells [dry weight] mol of glucose-1) for S. ruminantium HD4 grown in glucose-limited continuous culture.

Endogenous production of infectious Inoue-Melnick virus in a human meningioma cell line.

We investigated continuous production of Inoue-Melnick virus (IMV) in the MG-1 cell line, established from human meningioma. The infectious virus, identified as a type 1 virus, was mostly recovered extracellularly. Assay of MG-1 cells as infective centers indicated that most of the cells were capable of producing infectious virus. By immunofluorescence, more than 90% of the cells were found to have IMV-associated cytoplasmic antigen(s) (IMCA).

Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain

The growth of a denitrifying Pseudomonas strain on benzoic acid and 2-aminobenzoic acid (anthranilic acid) has been studied. The organism grew aerobically on benzoate, 2-aminobenzoate, and gentisate, but not on catechol or protocatechuic acid. These and other findings suggest that aerobic degradation of benzoic acid was via gentisic acid. Under completely anaerobic conditions in the presence of nitrate, benzoate and 2-aminobenzoate (5 mM each) were oxidized to CO2 with the concurrent reduction of NO3-to NO2-. Only after complete NO3-consumption was NO2-reduced to N2. Cells contained a NADP-specific 2-oxoglutaate dehydrogenase, in contrast to a NAD-specific pyruvate dehydrogenase. During anaerobic metabolism of [carboxyl-14C]benzoic acid, 16% of the label of metabolized benzoic acid was incorporated into cell material; this excludes intermediary decarboxylation during anaerobic metabolism. Extracts catalysed the activation of benzoic acid and a variety of its derivatives to the respective aryl-coenzyme A thioesters, ATP being cleaved to AMP and PPi; two synthetase activites were present. Extracts from 2-aminobenzoate-grown cells catalyzed a NADH-dependent reduction of 2-aminobenzoyl-CoA (100 nmol·min-1·mg-1 cell protein) to an unidentified CoA thioester, with a stoichiometric release of NH3 and a stoichiometry of ≈ 3 mol NADH oxidized per mol 2-aminobenzyol-CoA reduced when tested under aerobic conditions. The 2-aminobenzoyl-CoA reductase activity was lacking in anaerobic benzoate-grown cells and in aerobic cells. This is taken as evidence that 2-aminobenzoyl-CoA reductase is a key enzyme in a novel reductive pathway of anaerobic 2-aminobenzoic acid metabolism.

Uptake and release of glycine in the guinea pig cochlear nucleus.

This study attempts to determine if the cochlear nucleus (CN) contains glycinergic synaptic endings. The uptake and release of exogenous radiolabeled glycine were measured in vitro in the three major subdivisions of the guinea pig CN: anteroventral, posteroventral, and dorsal. A kinetic analysis of [3H]glycine uptake revealed the presence in each CN subdivision of a high- and a low-affinity uptake mechanism. The high-affinity mechanism had a Km of 25.2-30.5 microM and a Vmax of 3.8-4.8 nmol/10 mg of cell water/5 min, whereas the low-affinity mechanism had a Km of 633-718 microM and a Vmax of 26.6-37.1 nmol/10 mg of cell water/5 min. At steady state, the high-affinity mechanism accumulated 10 microM [3H]glycine from the medium, achieving tissue concentrations that were 13-24 times that in the medium. The high-affinity uptake was dependent on the temperature and on the concentrations of NaCl and glucose in the incubation medium. It exhibited a high degree of substrate specificity, as determined by the effects of structural analogues of glycine on the uptake of [3H]glycine. Each CN subdivision also contained two mechanisms mediating [14C]glycine release. One was activated by depolarizing electrical stimuli, produced a rapid transient release of [14C]glycine, and was dependent on the presence of extracellular Ca2+. The other was continuous, producing a slow spontaneous efflux of [14C]glycine. Released glycine could be removed primarily by uptake, because during release measurements, the amount of [14C]glycine detected in the medium decreased when glycine uptake activity was optimized. The electrically evoked, Ca2+-dependent release and the high-affinity uptake of glycine may mediate the synaptic release and inactivation of glycine, respectively. These findings, therefore, support the presence of glycinergic synaptic endings in each CN subdivision.

A simple method for isolation of lipopolysaccharides from Pseudomonas aeruginosa and some other bacterial strains.

A simple procedure for the isolation of bacterial lipopolysaccharides was developed. The method consists of the following three steps; recovery of lipopolysaccharides in an insoluble form together with denatured proteins in a hot MgCl2-Triton X-100 solution (step 1), solubilization with EDTA-Triton X-100 (step 2) and precipitation with MgCl2 (step 3). A good yield of lipopolysaccharides was obtained with most of the Pseudomonas strains tested. Although the yield was lower with strains belonging to Salmonella and Escherichia, irrespective of whether they are smooth-type or rough-type strains, the amounts obtained from 100mg of cells as dry weight were sufficient for detailed chemical characterization. The gel-electrophoretic profiles of the lipopolysaccharides prepared by the present method were essentially the same as those of the lipopolysaccharides prepared by the conventional phenol-water method. The method allowed us to prepare lipopolysaccharides from a large number of bacterial strains in one day.

Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate.

The binding of [125I]-human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0.7 nM and a binding capacity of 46 fmol hGH bound mg-1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH-stimulated leucine uptake by the cells in a time- and dose-dependent manner, half maximal effect being observed at 0.32 nM GH thus suggesting a direct relationship with the binding step.

Stereoselective uptake of the GABA-transaminase inhibitors gamma-vinyl GABA and gamma-acetylenic GABA into neurons and astrocytes.

The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomers R- and S-GVG and R- and S-GAG it could be shown that in both cell types only the S-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system for S-GVG (Km 78.2 +/- 20.3 microM; Vmax 0.71 +/- 0.06 nmol.min-1.mg-1 cell protein). In case of S-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes. S-GVG and S-GAG were found to be weak inhibitors of GABA uptake suggesting that S-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake of S-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T to S-GVG.

Contraction of collagen lattice by peritubular cells from rat testis.

Peritubular cells from 15- and 25-day-old rat testis trapped in collagen lattices caused those lattices to contract. Contraction proceeded more rapidly and to a greater extent using cells from younger rats. When 36,000 cells from 15- and 25-day-old rats were trapped in 800 mm2 lattices, the areas were reduced to 28 mm2 and 170 mm2, respectively, within 24 h. The cells from older rats were less effective at contracting the lattice than cells from younger rats. Cytochalasin B (5 micrograms ml-1) inhibited lattice contraction and caused disruption of actin filaments as seen by fluorescent staining with Rh-phalloidin. Cholera toxin (10 micrograms ml-1), and 1 mM-dibutyryl cAMP inhibited lattice contraction, as did 10 microM-trifluoperazine, commonly an inhibitor of calmodulin. The total intracellular concentration of cAMP was greater in peritubular cells from 25-day-old rats than in those from 15-day-old rats: 427 +/- 34 and 120 +/- 16 pmol mg-1 cell protein, respectively. When peritubular cells in monolayer were permeabilized with glycerol, the addition of ATP caused the cells to contract. Cell contraction was greater in cells from 15-day-old rats than 25-day-old rats. When cells were grown on silicone rubber, they caused that surface to wrinkle. Peritubular cells from 15-day-old rats caused the onset of wrinkling at 4 h. At the same time, no wrinkling was observed with cells from 25-day-old rats. Studies of lattice contraction and cell contraction were also made using cells from 20-day-old rats. In each case, contraction was intermediate between that of cells from 15-and 25-day-old rats. The possibility exists that lattice contraction, cell contraction and wrinkling of silicone film result from a mechanism of actin filament sliding, generated by myosin ATPase activity, and is inhibited by cAMP. The reduced rate of contraction in cells from 25-day-old rats may be related to their higher intracellular levels of cAMP. Evidence exists to show that cAMP blocks myosin ATPase activity by inhibiting the phosphorylation of its regulatory peptide, myosin light chain.