Total PKA Research Articles

Estimating occupational exposure in interventional radiology through Air Kerma Area Product - calculation of Effective Dose conversion factors

During interventional radiology procedures, interventionists are positioned close to the patient and directly exposed to ionizing radiation. This study presents a methodology to estimate the effective dose of interventionists through Air Kerma Area Product (PKA) in three interventional procedure modalities: coronary, cerebral, and peripheral. Optically stimulated luminescence dosimeters were used in several body regions (thyroid, eye lens, abdomen, feet, and hands) to assess equivalent doses and to convert external to internal doses – leading to effective doses related to each modality. These effective doses were then correlated to the total PKA given by the equipment. Thus, an effective dose estimate factor was calculated to represent the conversion between equipment parameters and the effective dose obtained from dosimeters, resulting in a novel methodology. The agreement between the methods was in the range of ± 0.13 μSv for angiographies and -8.3 to 5.3 μSv for angioplasties. These findings underscore the reliability of PKA as a predictor of effective dose, facilitating a rapid and precise assessment of occupational exposure in IR. This novel approach has the potential to enhance radiation safety protocols and optimize protection strategies for IR professionals, ensuring more accurate monitoring and control of radiation exposure in clinical settings.

Contribution of the fluoroscopy and cine modes to patient exposure in paediatric interventional cardiology procedures

IntroductionPaediatric interventional cardiology procedures have grown in recent years. The main disadvantage of these procedures is the high dose of radiation that patients receive. Such methods include the fluoroscopy mode, which allows real-time visualization, and the cine mode, which allows the documentation of images. The dosimetric quantities recommended currently for these procedures are: kerma-area product (PKA), air kerma at the patient entrance reference point (Ka,r), fluoroscopy time (tFl) and number of acquired images (IN). While PKA and Ka,r characterize total irradiation during the examination, IN and tFl describe the contribution of fluoroscopy and cine modes separately. These characteristics are indirect and presume that the radiation exposure is proportional to them. Such a choice is made because most of the equipment do not supply information about the contribution of fluoroscopy and cine modes to the PKA or Ka,r separately. ObjectivesThe primary objective of this work is to evaluate the contribution of the fluoroscopy and cine modes to the total exposure; we study to what extent these contributions can be characterized by fluoroscopy and cine time and number of acquired images. We also propose local diagnostic reference levels for quantities that separately describe the contribution of fluoroscopy and cine modes. MethodsIn a period of 3 years (2019–2021), data on 135 procedures were evaluated; the study included 53% of male patients (72) and 47% of female patients (63). Cardiac catheterization procedures were considered for diagnostic (59%) and therapeutic (41%) purposes. The average weight of patients was 21.4 ± 1.7 kg (2.0–112.0 kg), and the average age was 5.3 ± 0.4 years (0.0–17.1 years). Interventional cardiology procedures were performed using a GE Innova IGS 530 equipment (GE Healthcare, Milwaukee, WI, USA), manufactured in 2016 and installed in 2017, dedicated to the paediatric population. The necessary data on each procedure was extracted from the Radiation Dose Structure Reports, generated by the equipment at the end of the procedure. This report presents the PKA values for the fluoroscopy mode and the cine mode, total PKA, the fluoroscopy time, the cine time, and IN, as well as other data related to the procedure. The diagnostic reference levels (DRLs) proposed in this study were calculated as the 3-rd quartile of the corresponding distribution. ResultsThe contribution of the cine mode to the total kerma-area product is, on average, 50% though this mode lasted about 4% of the procedure time. Only the fluoroscopy time may be used among the indirect quantities to characterize the contribution of the fluoroscopy and cine modes. ConclusionsThe following local DRLs values are proposed: 2.0 Gy⋅cm2 for kerma-area product in fluoroscopy mode, 1.9 Gy⋅cm2 for kerma-area product in cine mode and 22 min (26 min for therapeutic procedures) for fluoroscopy time. For diagnostic and therapeutic procedures, the local DRLs values may be chosen as 1.5 Gy⋅cm2 and 2.5 Gy⋅cm2 correspondingly in both modes.

THE EFFECTS OF CONE-BEAM COMPUTED TOMOGRAPHY IMAGING GUIDANCE ON PATIENT RADIATION EXPOSURES IN TRANS-ARTERIAL CHEMOEMBOLISATION FOR HEPATOCELLULAR CARCINOMA

This study investigated the effects of cone-beam computed tomography (CBCT) guidance in trans-arterial chemoembolisation (TACE) procedures on the number of digital subtraction angiography (DSA) runs acquired and total patient radiation exposure in patients with hepatocellular carcinoma (HCC). A retrospective, analytical cross-sectional, single institution, study was conducted. Dose data were compared across the control (DSA guidance alone) and study (DSA and CBCT guidance) groups. A total of 122 procedures were included within the study. There was a significant reduction in the number of DSA runs (3 vs 5, p < 0.001) and DSA air kerma-area product (PKA) (3077.3 vs 4276.6 μGym2, p = 0.042) for the study group when compared to the control group. Total procedural PKA and total procedural reference air kerma (Ka,r) were shown to be 50 and 73% higher, respectively, for the study group when compared to the control group. CBCT imaging guidance does reduce the number of DSA runs and DSA PKA required to complete the TACE procedure for patients diagnosed with HCC; however, a substantial increase in total procedural PKA is to be expected and it is thus important that this increased dose is carefully considered and justified.

Effects of progesterone nanoparticles on the sperm capacitation and acrosome reaction in asthenozoospermia men.

Low motility is one of the causes of male infertility. In this study, the effects of progesterone solid lipid nanoparticles (SLNs) on sperm capacitation, acrosome reaction, oxidative stress and expression of SPACA1 and MAPK way genes were investigated. Progesterone SLNs were synthesized using the solvent emulsification evaporation method. Twenty asthenozoospermia samples were selected, and sperm and acrosome membrane integrity, acrosome reaction, sperm motility, viability, total antioxidant capacity (TAC), total oxidative status tests and PKA, PTK, P38MAPK and SPACA1 gene expressions were assessed. The synthesized nanoparticles were prepared with the size (187.6nm), PDI (0.184), EE (85.82%), LP (3.43%) and ZP (-23.5mV). Progesterone SLNs increased sperm and acrosome membrane integrity and TAC (p<.05). Also, the expression of P38MAPK, PKA, PTK, and SPACA1 genes in this group showed a significant increase (p<.001). Progesterone SLNs increased acrosome reaction, sperm capacitation and TAC. Also, it increased the expression of PTK PKA, SPACA1 and P38MAPK genes.

Glucagon Reduces Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice.

Sepsis is one of the most common comorbidities observed in diabetic patients, associated with a deficient innate immune response. Recently, we have shown that glucagon possesses anti-inflammatory properties. In this study, we investigated if hyperglucagonemia triggered by diabetes might reduce the migration of neutrophils, increasing sepsis susceptibility. 21 days after diabetes induction by intravenous injection of alloxan, we induced moderate sepsis in Swiss-Webster mice through cecum ligation and puncture (CLP). The glucagon receptor (GcgR) antagonist des-his1-[Glu9]-glucagon amide was injected intraperitoneally 24h and 1h before CLP. We also tested the effect of glucagon on CXCL1/KC-induced neutrophil migration to the peritoneal cavity in mice. Neutrophil chemotaxis in vitro was tested using transwell plates, and the expression of total PKA and phospho-PKA was evaluated by western blot. GcgR antagonist restored neutrophil migration, reduced CFU numbers in the peritoneal cavity and improved survival rate of diabetic mice after CLP procedure, however, the treatment did no alter hyperglycemia, CXCL1/KC plasma levels and blood neutrophilia. In addition, glucagon inhibited CXCL1/KC-induced neutrophil migration to the peritoneal cavity of non-diabetic mice. Glucagon also decreased the chemotaxis of neutrophils triggered by CXCL1/KC, PAF, or fMLP in vitro. The inhibitory action of glucagon occurred in parallel with the reduction of CXCL1/KC-induced actin polymerization in neutrophils in vitro, but not CD11a and CD11b translocation to cell surface. The suppressor effect of glucagon on CXCL1/KC-induced neutrophil chemotaxis in vitro was reversed by pre-treatment with GcgR antagonist and adenylyl cyclase or PKA inhibitors. Glucagon also increased PKA phosphorylation directly in neutrophils in vitro. Furthermore, glucagon impaired zymosan-A-induced ROS production by neutrophils in vitro. Human neutrophil chemotaxis and adherence to endothelial cells in vitro were inhibited by glucagon treatment. According to our results, this inhibition was independent of CD11a and CD11b translocation to neutrophil surface or neutrophil release of CXCL8/IL-8. Altogether, our results suggest that glucagon may be involved in the reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. This work collaborates with better understanding of the increased susceptibility and worsening of sepsis in diabetics, which can contribute to the development of new effective therapeutic strategies for diabetic septic patients.

Concentration Addition, Independent Action, and Quantitative Structure-Activity Relationships for Chemical Mixture Toxicities of the Disinfection By products of Haloacetic Acids on the Green Alga Raphidocelis subcapitata.

The potential toxicity of haloacetic acids (HAAs), common disinfection by products (DBPs), has been widely studied; but their combined effects on freshwater green algae remain poorly understood. The present study was conducted to investigate the toxicological interactions of HAA mixtures in the green alga Raphidocelis subcapitata and predict the DBP mixture toxicities based on concentration addition, independent action, and quantitative structure-activity relationship (QSAR) models. The acute toxicities of 6 HAAs (iodoacetic acid [IAA], bromoacetic acid [BAA], chloroacetic acid [CAA], dichloroacetic acid [DCAA], trichloroacetic acid [TCAA], and tribromoacetic acid [TBAA]) and their 68 binary mixtures to the green algae were analyzed in 96-well microplates. Results reveal that the rank order of the toxicity of individual HAAs is CAA > IAA ≈ BAA > TCAA > DCAA > TBAA. With concentration addition as the reference additive model, the mixture effects are synergetic in 47.1% and antagonistic in 25%, whereas the additive effects are only observed in 27.9% of the experiments. The main components that induce synergism are DCAA, IAA, and BAA; and CAA is the main component that causes antagonism. Prediction by concentration addition and independent action indicates that the 2 models fail to accurately predict 72% mixture toxicity at an effective concentration level of 50%. Modeling the mixtures by QSAR was established by statistically analyzing descriptors for the determination of the relationship between their chemical structures and the negative logarithm of the 50% effective concentration. The additive mixture toxicities are accurately predicted by the QSAR model based on 2 parameters, the octanol-water partition coefficient and the acid dissociation constant (pKa ). The toxicities of synergetic mixtures can be interpreted with the total energy (ET ) and pKa of the mixtures. Dipole moment and ET are the quantum descriptors that influence the antagonistic mixture toxicity. Therefore, in silico modeling may be a useful tool in predicting disinfection by-product mixture toxicities. Environ Toxicol Chem 2021;40:1431-1442. © 2021 SETAC.

Whole-Cell cAMP and PKA Activity are Epiphenomena, Nanodomain Signaling Matters.

Novel targeted fluorescent biosensors provide key insights into very local nanodomains of cAMP and PKA activity, and how they respond differently to β-adrenergic activation in cardiac myocytes. This unique spatiotemporal detail in living cells is not available with biochemical measurements of total cellular cAMP and PKA, and provides unique physiological insights.

DFT methods applied to answer the question: how accurate is the ligand acidity constant method for estimating the pKa of transition metal hydride complexes MHXL4 when X is varied?

Density functional theory (DFT) is used to calculate the relative free energies of deprotonation of the isomers of iron-group hydride complexes MHXL4 where M = Fe, Ru, Os, L4 = (CO)4 or (PMe2CH2CH2PMe2)2 for a wide range of anionic ligands X. The free energies of the most stable isomers are used to calculate relative pKa values where Ka refers to the acid dissociation constant for the equilibrium MHXL4 → [MXL4]- + H+. These are used to test the proposal that the pKa for a given metal complex in THF can be simply calculated by adding the contributions to the total pKa value from each ligand L; these are called ligand acidity constants (LAC) AL used in the LAC equation [R. H. Morris, J. Am. Chem. Soc., 2014, 136, 1948-1959]. The AL are calculated using AL = 0.2 as a reference for the hydride ligand. The AL of certain less polarizable X ligands are found to be fairly constant (±1 to ±2 units) and consistent with the proposed LAC method for a range of the complexes considered: 2 for Me-, 1 for OH-, 1 for NH2-, 0 for B(OCH2CH2O-)-, -1 for OMe-, -2 for SH- and -2 for SMe-. Other ligands have more variable AL values (±3 to ±5 units) because of high polarizability or other reasons: 1 for OtBu-, -1 for F-, -2 for BMe2-, -2 for NMe2-, -3 for Cl-, -3 for PMe2-, -3 for Br-, -5 for I-, -6 for CN- and -12 for SiCl3-. Iodide stabilizes the anion [MIL4]- more than does fluoride in [MFL4]- making iodide the more acidifying ligand despite its lower electronegativity. DFT is also used to validate the charge correction in the LAC equation.

Radiation exposure to patients and medical staff in hepatic chemoembolisation interventional procedures in Recife, Brazil.

The purpose of this study was to evaluate patient and medical staff absorbed doses received from transarterial chemoembolisation of hepatocellular carcinoma, which is the most common primary liver tumour worldwide. The study was performed in three hospitals in Recife, capital of the state of Pernambuco, located in the Brazilian Northeastern region. Two are public hospitals (A and B), and one is private (C). For each procedure, the number of images, irradiation parameters (kV, mA and fluoroscopy time), the air kerma-area product (PKA) and the cumulative air kerma (Ka,r) at the reference point were registered. The maximum skin dose (MSD) of the patient was estimated using radiochromic film. For the medical staff dosimetry, thermoluminescence dosemeters (TLD-100) were attached next to the eyes, close to the thyroid (above the shielding), on the thorax under the apron, on the wrist and on the feet. The effective dose to the staff was estimated using the algorithm of von Boetticher. The results showed that the mean value of the total PKA was 267.49, 403.83 and 479.74 Gy cm(2) for Hospitals A, B and C, respectively. With regard to the physicians, the average effective dose per procedure was 17 µSv, and the minimum and maximum values recorded were 1 and 41 µSy, respectively. The results showed that the feet received the highest doses followed by the hands and lens of the eye, since the physicians did not use leaded glasses and the equipment had no lead curtain.

The lateral plane delivers higher dose than the frontal plane in biplane cardiac catheterization systems.

The objective of the study is to compare radiation dose between the frontal and lateral planes in a biplane cardiac catheterization laboratory. Tube angulation progressively increases patient and operator radiation dose in single-plane cardiac catheterization laboratories. This retrospective study captured biplane radiation dose in a pediatric cardiac catheterization laboratory between April 2010 and January 2014. Raw and time-indexed fluoroscopic, cineangiographic and total (fluoroscopic + cineangiographic) air kerma (AK, mGy) and kerma area product (PKA, µGym(2)/Kg) for each plane were compared. Data for 716 patients were analyzed: 408 (56.98 %) were male, the median age was 4.86 years, and the median weight was 17.35 kg. Although median beam-on time (minutes) was 4.2 times greater in the frontal plane, there was no difference in raw median total PKA between the two planes. However, when indexed to beam-on time, the lateral plane had a higher median-indexed fluoroscopic (0.75 vs. 1.70), cineangiographic (16.03 vs. 24.92), and total (1.43 vs. 5.15) PKA (p < 0.0001). The median time-indexed total PKA in the lateral plane is 3.6 times the frontal plane. This is the first report showing that the lateral plane delivers a higher dose than the frontal plane per unit time. Operators should consciously reduce the lateral plane beam-on time and incorporate this practice in radiation reduction protocols.

Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters

Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.

Effective solution chemistry instruction by integrating live pC–pH diagrams into PowerPoint

AbstractpC–pH diagrams provide a graphical illustration of chemical speciation as a function of pH and are hence a very effective tool for understanding the chemical system being studied. This understanding can lead to assumptions that simplify problem solving. However, manual construction of pC–pH diagrams for multiprotic systems is cumbersome and is further complicated by non‐standard temperature and ionic strength conditions. As a result, construction of pC–pH diagrams can become the focus of classroom teaching rather than their application to solving real‐world problems. Students tend to get lost in the complexities of pC–pH diagram construction and often fail to appreciate their value in problem solving. To address this limitation, we have developed interactive pC–pH diagrams for mono, di, and triprotic systems that are all bundled into a single PowerPoint file. The governing equilibrium equations are presented adjacent to the pC–pH diagram which makes the entire process transparent. The diagrams update in real‐time when the total concentration and pKa are changed by the user making them universally applicable to any mono, di, or triprotic system. Temperature impact on pKa and the pC–pH diagram can also be examined for some commonly encountered systems. Because of PowerPoint integration, there is no learning curve and specification of pKa is typically all that is required to use these diagrams. A real‐world application of these interactive pC–pH diagrams is presented by examining the solution chemistry in therapeutic protein‐producing mammalian cell bioreactors. The cause of high bioreactor CO2 concentration and strategies to overcome it were determined just by using the interactive pC–pH diagrams without resorting to numerical calculations. We believe these easy to use and interactive pC–pH diagrams could be more extensively used for problem solving in the classroom simultaneously enhancing student understanding of the chemical system being studied. The PowerPoint file with the pC–pH diagrams is available free of charge for academic use. © 2010 Wiley Periodicals, Inc. Comput Appl Eng Educ 20: 741–749, 2012

Depressed PKA Activity Contributes to Impaired SERCA Function and is Linked to the Pathogenesis of Glucose-induced Cardiomyopathy

K. Dutta, M. W. Carmody, S. E. Cala and A. J. Davidoff. Depressed PKA Activity Contributes to Impaired SERCA Function and is Linked to the Pathogenesis of Glucose-induced Cardiomyopathy. Journal of Molecular and Cellular Cardiology (2002) 34, 985–996. We have previously described a cardiomyopathy induced by culturing ventricular myocytes from normal adult rats in a medium containing high concentrations of glucose, which recapitulates cellular changes associated with early onset diabetic cardiomyopathy. This investigation was designed to evaluate cellular mechanisms that could contribute to slowed cytosolic Ca2+ removal and myocyte relaxation in glucose-induced cardiomyopathy. Isolated ventricular myocytes were cultured overnight in medium containing normal glucose (n=5.5mM) or high glucose (HG=25.5mM). Cytosolic Ca2+ removal was monitored with fluo-3 and myocyte mechanics with video-edge detection. Electrically stimulated Ca2+ transients were prolonged in HG cells (AT/PK=215±7ms, n=41) compared to N myocytes (AT/PK=173±5ms, n=34). By pharmacological and ionic manipulations, Ca2+ removal attributable to SERCA was slower in the HG group (AD/PK=290±17ms,n =41) compared to N (AD/PK=219±10, n=34), whereas NCX function was similar in both groups of cells. Total PKA activity was depressed in HG myocytes by 56% compared to N cells. β-adrenergic receptor stimulation with ISO (10−7M) normalized myocyte relaxation, Ca2+ transients and PKA activity in HG myocytes. Furthermore, inhibition of PKA with H89 (10−5M) depressed peak fractional shortening (PS) and slowed relengthening (AR/PK) to a greater extent in N (−50% for PS and 92% for AR/PK) than in HG cells (−25% for PS and 48% AR/PK). Depressed cytosolic Ca2+ removal was not, however, associated with changes in basal levels of phosphorylated PLB, nor levels of SERCA, NCX or PLB proteins. We conclude that cellular mechanisms associated with the early onset glucose-induced cardiomyocyte dysfunction involves alterations in Ca2+ regulation, which may be a common manifestation of other forms of cardiomyopathies.

Expression of enzymes of covalent protein modification during regulated and dysregulated proliferation of mammary epithelial cells: PKA, PKC and NMT.

Three proteins are functionally interlinked in the targeting of protein phosphorylation catalyzed by the C-subunit of PKA: PKA itself, AKAPs and NMT. Furthermore, in a variety of biological contexts, mechanisms exist whereby PKA and PKC are able to modulate the activity of one another. We have investigated the expression and subcellular distribution of these proteins in two models of mammary cell proliferation and differentiation--the normal rat mammary gland during pregnancy and lactation and human breast tissue before and after malignant transformation. Modulation of PKA does not acutely affect activity or sub-cellular distribution of PKC in mammary acini, nor does modulation of PKC acutely affect PKA activity or subcellular distribution. Therefore, the co-ordinate expression of these two protein kinases in normal and cancerous mammary epithelial cells and the greater basal activation level of them both accompanying increased mitogenic activity, which we have reported, does not result from short-term cross-talk between them. Although basal and total levels of PKA diminish in rodent mammary epithelial cells during the transition from proliferative to secretory functional mode, the level of expression of AKAPs increases. The expression of two apparently mammary-specific and mostly membrane-associated AKAPs is tightly linked to the onset and maintenance of differentiated function in rat mammary tissue. Paradoxically, the probable analogues of these two AKAPs in human mammary tissue are hyperexpressed when normal epithelial cells transform to a cancer phenotype--conventionally regarded as a process involving a degree of dedifferentiation. Mammary AKAP hyperexpression in breast cancers is accompanied by increases in the levels of total and basal PKA. One mechanism whereby NMT is targeted to membranes, via interaction with ribosomal proteins, has recently been elucidated. Our data support the contention that the localization of NMT is an important variable in the regulation of cellular proliferation, but they do not characterize the mechanisms whereby the differential targeting of NMT is achieved. As yet we lack a full tool-kit with which to examine NMT either to draw firm conclusions regarding the identity of particular isoforms found in particular sub-cellular locations or to define the relationships between these different molecular variants. However, it is technically possible to transfect cells with inducible NMT expression constructs engineered in such a way that the recombinant, catalytically competent, NMT that they encode is targeted either to membranes or to cytosol: an exploration of the effects of such transfections on cellular proliferation would afford a critical test of the mechanistic involvement of NMT in the control of mitogenesis.

Measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled Kemptide

Traditional protein kinase assays include the use of [32P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 +/- 0.8% and 14.3 +/- 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20-inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.

Neuronal cAMP-dependent protein kinase type II is concentrated in mushroom bodies of Drosophila melanogaster and the honeybee Apis mellifera.

In both Drosophila melanogaster and the honeybee Apis mellifera, cyclic adenosine monophosphate (cAMP)-dependent processes have been implicated in mechanisms of learning. This study characterizes the type II cAMP-dependent protein kinase (PKAII), the major target of cAMP in adult animals. In both species, PKAII is restricted to neuronal tissue, in which it accounts for more than 90% of total PKA activity. Although the intensity of PKAII immunoreactivity differs between distinct brain regions, labeling is detectable in all neuropiles and most somata. While the visual neuropiles, the antennal lobes, and structures of the central brain exhibit intermediate immunostaining, the mushroom bodies show high labeling and contain a three- to fourfold higher PKA activity compared to other neuropiles. Since the mushroom bodies are central sites of olfactory learning mediated via cAMP-dependent signaling, the modulatory functions of transmitters on PKA activity in Kenyon cells from the honeybee were tested. Agents which elevate cytoplasmic Ca2+ levels have no effects on PKA activity in cultured Kenyon cells. Dopamine, serotonin, and octopamine, however, cause an increase in PKA activity in Kenyon cells. The modulation of PKA activity by octopamine, the putative transmitter of the unconditioned stimulus in associative olfactory learning in the honeybee, together with the findings on the central role of the cAMP cascade in Drosophila mushroom bodies, suggests a major implication of PKAII-mediated phosphorylation in learning and memory in both Drosophila and Apis.

Buffer effects on aqueous swelling kinetics of polyelectrolyte gels

AbstractElectrolytes are often added to a gel‐swelling medium under the assumption that the important conditions which characterize swelling rates are the solution pH and ionic strength, with little emphasis on the nature of the electrolyte. Previous research by Siegel et al. has indicated that the presence of the un‐ionized acidic form of an electrolyte buffer is a primary rate determinant for swelling of a polybase gel. A systematic swelling study on two separate gels, 2‐hydroxyethyl methacrylate copolymerized with methacrylic acid (HEMA/MAA) and N,N‐dimethylaminoethyl methacrylate (HEMA/DMA), has been performed to investigate the influence of the concentration of the un‐ionized buffer by three principal factors: (1) total buffer concentration, (2) solution pH, and (3) buffer pKa. Swelling and deswelling kinetics were obtained. In the presence of an electrolyte buffer, a dramatic swelling rate increase is observed for the HEMA gels, with substantial gains in rate obtained as total buffer concentration rises. Results also emphasize that to enhance swelling kinetics, the pH must be such that the buffer is essentially un‐ionized.

Changes in cAMP-dependent and Ca 2+-phospholipid-dependent protein kinase activities in suspension cultures of porcine thyroid cells

The morphological and functional characteristics and the activities of cyclic AMP- (PKA I and PKA II) and calcium and phospholipid-dependent (PKC) protein kinases were studied in 2-day-old suspension cultures of porcine thyroid cells and were compared with those in freshly dissociated cells and intact glands. Thyroid cell morphology changed during the 2-day culture in the absence of specific regulators. This is characterized by a loss of cellular polarity, exo- and endocytotic vesicles and membranes of the rough endoplasmic reticulum, and an increase in the number of lysosomes, pseudomyelinic structures, lipidic inclusions and free ribosomes. Functional changes are characterized by a progressive decrease in protein iodination and its sensitivity to TSH stimulation. The total PKA activity in the cytosols of these cultures was slightly greater than that of freshly prepared tissue, due to the selective and significant accumulation of PKA I in cultured cells. In the particulate fraction the PKA activity was unchanged. PKC is the major kinase activity in porcine thyroids, and remains so in cultured cells. The slight drop in its activity in cytosols was offset by a significant increase in the particulate fraction, suggesting an intracellular redistribution of this kinase in cultured cells. The PKC activity is also partly activated in both the cytosol and particulate fraction, which results in an increased basal activity. The changes in PKA and PKC activities greatly modified the PKC/PKA ratios in the cytosols and the particulate fractions of cultured cells. These modifications could be partly responsible for the changes in sensitivity of cultured cells to the agents which control their activity.

Thyrotropin but not epidermal growth factor down-regulates the isozyme I (PKa I) of cyclic AMP-dependent protein kinases in dog thyroid cells in primary cultures

The activity of the two cAMP-dependent protein kinases (PKa I and PKa II) was evaluated in dog thyroid cells in primary cultures after a 6-day growth period induced by either thyrotropin (TSH) or epidermal growth factor (EGF). Although the total PKa activity was not affected in cells cultured in the presence of TSH or EGF, their actions on the PKa I and PKa II expressions were significantly different. The activity of PKa I was strongly inhibited by TSH (70–80%) while with EGF it was either stimulated or unaffected with respect to controls. The two mitogens did not have a significant effect on the activity of PKa II. Forskolin (Fk) mimicked the effect of TSH. The expression of the two regulatory subunits (R I and R II), evaluated by the covalent binding of 8-azido-cAMP, was similar to the expression of the corresponding catalytic activities, suggesting a coregulation of the catalytic and regulatory subunits from the same isozyme. After chronic stimulation by TSH, differentiated dog thyroid cells are almost completely deprived of PKa I.