Vanadate and brain adenylate cyclase. effect of spreading depression

The effect of vanadate on the adenylate cyclase activity of rat cerebral cortex homogenates is described. In the absence of ethyleneglycol-bis-(β-aminoethyl ether) N,N'-tetraacetic acid (EGTA), 10^?6 M vanadate inhibited enzyme activity by 23%, while 10^?4 M and 10^?3 M stimulated the enzyme by 14 and 90%, respectively. In the presence of 0.2 mM EGTA, 10^?6 M to 10^?3 M vanadate had only stimulating effects (18–450%). Additive effects of vanadate and noradrenaline on adenylate cyclase activity suggest different sites of action of these agents. Interaction of vanadate with both fluoride and guanyl-5'-yl imidodiphosphate had an apparently competitive character. Adenylate cyclase maximally stimulated by fluoride (10 mM) was inhibited by vanadate. This inhibitory effect was more pronounced in the absence of EGTA. Adenylate cyclase in the homogenates from the rat cerebral cortex in vivo invaded by spreading depression was slightly increased (up to 38%). This effect was abolished by low (10^?7 M) vanadate.The results suggest that brain adenylate cyclase is stimulated by vanadate via the guanine nucleotide regulatory protein. The mechanism of vanadate's action, its modulation by calcium ions and the possible physiological role of these effects are discussed.     

Effect of adrenergic compounds, aminophylline and hydrocortisone, on in vitro immunoglobulin synthesis by normal human peripheral lymphocytes

Suspensions of normal human peripheral lymphocytes were exposed briefly to various concentrations of propranolol, isoproterenol, epinephrine, or aminophylline (both without and with added hydrocortisone) and then incubated for 72 hours at 37 ° C. After this incubation the amount of immunoglobulin (Ig) synthesized and secreted into the cultured supernatants was quantitated by radiaimmunoassay. Significantly increased Ig formation compared to controls was observed with the following concentrations of the experimental compounds (without hydrocortisone): 10^?9 to 10^?7M propranolol, 10^?10 to 10^?7M isoproterenol, 10^?8M epinephrine, and 10^?7 to 10^?5M aminophylline. When cell suspensions were exposed briefly to these same compounds but with hydrocortisone added, the following concentrations of compounds significantly increased Ig synthesis: 10^?10 to 10^?8M propranolol, 10^?10 to 10^?6M isoproterenol, 10^?9 to 10^?7M epinephrine, and 10^?8 to 10^?5M aminophylline. Of the four compounds studied, only the epinephrine data suggest that hydrocortisone enhanced the stimulation of Ig synthesis by this catecholamine.     

Inhibition of ACTH-induced differentiation of cortical cells and their mitochondria by corticosterone in tissue culture of fetal rat adrenals

Tissue cultures of fetal rat adrenals were used to study the effects of corticosterone on the ACTH-induced ultrastructural differentiation of cortical cells and their mitochondria. Corticosterone in dosages of 0.2, 2.0, 5.0, 10, and 20 μg/ml (corresponding to concentrations of 6 × 10^?7, 6 × 10^?6, 1.5 × 10^?5, 3 × 10^?5, and 6 × 10^?5 molar) was added alone or together with 100 mU/ml of ACTH to the culture medium, daily from the sixteenth day of cultivation up to and including the twenty-first day. Corticosterone alone induced no ultrastructural changes in cortical cells. Corticosterone in concentrations of 6 × 10^?7 to 3 × 10^?5 M given with ACTH induced hypertrophy of Golgi apparatus. Corticosterone in concentrations of 6 × 10^?5 M inhibited the ACTH-induced differentiation of cortical cells. However, the nuclear chromatin increased and Golgi apparatus was strikingly hypertrophied. Mitochondria often aggregated adjacent to the nuclear envelope but their ultrastructure remained undifferentiated with tubular or tubulovesicular cristae. Ribosomes appeared as single particles. A marked increase of smooth surfaced endoplasmic reticulum was noted also in cortical cells treated with 6 × 10^?5 M of corticosterone. The present observations suggest that corticosterone acts as an intracellular inhibitor in cortical cells. It appears to inhibit cytoplasmic protein synthesis at the ribosomal level and prevents synthesis of cytoplasmic mitochondrial protein synthesis stimulating factor and the latter, in turn, inhibits the activation of mitochondrial protein synthesis. A new model is presented to explain the regulation of growth and secretion in the adrenal cortex.     

Influence of Sublethal Antibiotic Concentrations on Bacterial Adherence to Saliva-Treated Hydroxyapatite

The influence of growth in the presence of sublethal concentrations of nine antibiotics on the ability of certain potentially odontopathic bacteria to attach to saliva-treated hydroxyapatite surfaces which mimic teeth was studied. Cells of Actinomyces viscosus LY7 and S2, Bacteroides gingivalis 381, Capnocytophaga ochraceus 6, and Actinobacillus actinomycetemcomitans N27 attached in lower numbers to saliva-treated hydroxyapatite when grown in the presence of 50% of the minimum inhibitory concentration of tetracycline. Electron microscopic observations of negatively stained preparations indicated that tetracycline-grown A. viscosus LY7 cells had fewer fimbriae than did untreated cells, which may account for the impaired ability of the treated cells to attach. However, cells of Actinomyces naeslundii L13 and S4 attached in higher numbers when grown in the presence of tetracycline, clindamycin, erythromycin, chloramphenicol, or neomycin. Streptococcus mutans strains H12 and JBP also exhibited increased adherence to saliva-treated hydroxyapatite when grown in the presence of 50 or 25% of the minimum inhibitory concentration of penicillin. Thus, growth in the presence of sublethal antibiotic concentrations could increase as well as decrease the adherence of bacteria to saliva-treated hydroxyapatite. Antibiotic-grown cells of the Actinomyces strains showed enhanced hemagglutination activity, but this did not correlate with their ability to attach to saliva-treated hydroxyapatite. Sublethal concentrations of antibiotics in the growth media also affected the coaggregation reactions of several organisms; the effects were specific for one member of the coaggregation pair.     

Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10^?8) than in biofilm (9.9 × 10^?9) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10^?9 in the planktonic mode to 6 × 10^?8 in biofilm, whereas normomutators (from 9.4 × 10^?8 to 5.3 × 10^?8) and hypermutators (from 1.6 × 10^?6 to 7.7 × 10^?7) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.     

Components coupling cholinergic excitation with contraction of smooth muscles of guinea pig taenia coli

Acetylcholine (AC) in concentrations of 10^?9–10^?7 g/ml increases the smooth-muscle tone of the guinea pig taenia coli by increasing the permeability of the cell membranes to inward flows of Ca⁴⁵ ions. In concentrations of 10^?6 g/ml or higher AC induces the liberation of membrane calcium and, in concentrations of 10^?5–10^?3 g/ml, it significantly increases the membrane permeability of the smooth-muscle cells to Na²² ions, causing depolarization and an increase in action potential frequency. It is postulated that the mechanism coupling the cholinergic stimulus with the final effect (muscle contraction) includes three components: increased entry of Ca⁺⁺ into the smooth-muscle cells, liberation of membrane calcium, and a spike mechanism.     

Mechanism of the polymerization of acrylamide initiated by the glycerol/Ce(IV) redox system

The mechanism of the polymerization of acrylamide in aqueous medium initiated by the glycerol (R)/Ce(IV) redox system was studied. The rate of monomer disappearance was found to be directly proportional to [M]^3/2, and R^1/2 at lower concentrations of Ce(IV). The rate of the disappearance of ceric ions was found to be inversely proportional to [M] and directly proportional to [Ce(IV)] and [R]. Consistent with the findings of earlier investigations, a complex formation between monomer, acrylamide, and ceric ion is indicated. The experimental results show the termination to be mutual. On the basis of these and other kinetic results, an appropriate mechanism is proposed. At higher concentration of Ce(IV), a different mechanism seems to operate. The rates of disappearance of both the monomer and ceric ions are retarded on addition of anions like HSO₄^?, SO₄^{? ?}, or CIO₄^?, but they are accelerated on the addition of Mn(II) ions. Interpretations of the above observations are furnished.     

17β‐Estradiol Exposure Accelerates Skeletal Development in Xenopus laevis Tadpoles

Although it is well established that estrogen regulates skeletal growth and ossification in mammals, the effects of estrogen on skeletal development in amphibians are relatively uncharacterized. This study was conducted to characterize the impact of 17β‐estradiol exposure on skeletal development in Xenopus laevis tadpoles. On day 48 postfertilization, tadpoles were placed in tanks containing 50% Holtfreter's Solution ±17β‐estradiol at one of four concentrations (10^?11, 10^?10, 10^?9, and 10^?8 M). At 7–11 day intervals until day 91, 7–10 tadpoles per group were killed, fixed, measured, and staged. Specimens were then cleared and double‐stained for cartilage and bone, and 34 skeletal elements were analyzed for ossification. Results from the study indicate that both low (10^?11 M) and high (10^?8 M) concentrations of 17β‐estradiol have a significant stimulatory effect on tadpole development. Both the larval stage and ossification index of tadpoles exposed to 10^?11 or 10^?8 M 17β‐estradiol were significantly greater than those observed in control animals by day 91 postfertilization. These results are consistent with the hypothesis that endogenous and exogenous estrogen could play a role in the regulation of bone ossification in amphibians. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Visualization of serotonin neurones in the nodose ganglia of the cat. An autoradiographic study

After in vitro incubation of the nodose ganglia (right and left) in tritiated serotonin mixed with norepinephrine, the autoradiographic detection of the amine at the light-microscopic level showed an intense labeling over cell bodies; they have been characterized as gray type neurones at the ultrastructural level. Moreover, a labeling was observed over unmyelinated intraganglionic fibers.The uptake of exogenous serotonin by these neurones was demonstrated to be specific by using different experimental conditions. The same distribution of labeling was observed either after the incubation in ‘high affinity’ concentration (10^?7M), or in saturated concentrations (10^?5M or 10^?4M) of [³H]serotonin. The number of labeled cell bodies decreased after adding fluoxetine (an inhibitor of serotonin uptake) to the tracer.The specificity of the neuronal accumulation of serotonin was confirmed by interganglionic comparisons between the nodose and the superior cervical ganglia incubated under the same conditions. Several hundred cell bodies were labeled in the nodose ganglion incubated in [³H]serotonin (10^?6M) whereas there was no labeling in the superior cervical ganglion. On the other hand, most of the perikarya were labeled in the superior cervical ganglion incubated in [³H] norepinephrine (10^?6M), whereas no significant labeling was found in the nodose ganglion.These results provide some evidence that peripheral sensory neurones are able to take up exogenous serotonin.     

Effects of pro-opiomelanocortin fragments on release of catecholamines from adrenal chromaffin cells

The effects of pro-opiomelanocortin (POMC) peptide fragments on the basal and agonist-induced release of catecholamines (CAs) from monolayer cultures of purified bovine adrenal chromaffin cells were tested. None of the 5 peptides tested, i.e. ß-MSH, ACTH_1–39, γ-MSH_1–13. γ₃-MSH and N-terminal POMC fragment, had any effect on basal CA release. However, ß-MSH (10^?5 M), γ-MSH_1–13 (10^?6–10^?5 M), γ₃-MSH (10^?5 M) and N-terminal POMC fragment (10^?5 M) inhibited the nicotine-induced release of CAs from the chromaffin cells. The possible physiological significance of this inhibitory neuromodulation is discussed.     

Inhibition of axoplasmic transport in the developing visual system of the rat—I. Structural changes in the retina and optic nerve with graded doses of intraocular colchicine

In order to study the role of axonal transport in the mediation of transneuronal metabolic stimulation upon a population of differentiating neurons, colchicine, a potent inhibitor of rapid and slow phases of axonal transport, was injected into the eye of albino rats at 1,3,5, 10, 15 and 20 days postnatal in concentrations ranging from 10^?5 M to 2 × 10²M and in quantities of 0.3 to 0.5μl. Quantitative light and electron microscopy were subsequently employed to assess reactive alterations in the developing retina and optic nerve. Application of colchicine severely retarded the development of the sensory elements, with disappearance of synaptic ribbons of sensory cell axons, a significant reduction in the thickness of the inner plexiform layer, due to the presence of numerous shrunken synaptic elements and the appearance of rosettes of sensory cells displaced to the inner nuclear layer. These alterations were found to be dose-dependent. Counts of ganglion cell populations at various times after application of colchicine demonstrated optimal concentrations which could be injected at each postnatal age without causing ganglion cell degeneration. Ultrastructural examination of such cells revealed varying degrees of disorganization and dissolution of the endoplasmic reticulum with the formation of occasional small cytoplasmic vacuoles. Higher concentrations of colchicine caused extensive vacuole formation in all classes of retinal neurons, scattered hyperchromic cells and widespread degeneration and autolysis.The diameter of the optic nerve was reduced to 60–95% of normal following intraocular colchicine. depending on the concentration employed, but electron microscopy revealed normal patterns of distribution of axoplasmic microtubules and filaments in control and experimental animals and quantitative analysis revealed no significant loss of axons. While no reactive changes took place in individual elements, the periphery of the nerve was often indented by a highly-folded glia limitans.Maximal doses of intraocular colchicine for each age level were established by this study. These were: 1 day, 10^?3 M: 5 days, 5 × 10^?3M; 10 days, 5 × 10^?3M; 15 and 20 days, 10^?2 M. The information derived from this morphological analysis provides the foundation for subsequent measurements of axonal transport inhibition in the developing visual system to be reported in the second article of this series.     

Effect of adrenalin,noradrenalin, dopamine,dopa, and phenylalanine on peroxidation of lipids in liver mitochondrial membranes

The effect of catecholamines on chain peroxidation of lipids in the liver mitochondrial membranes in the presence of Fe⁺⁺ ions was studied by a very weak chemiluminescence method. Catecholamines between concentrations of 10^?6 and 10^?4 M were shown to inhibit this process. By a mathematical investigation of the kinetics of the process constants of antioxidative activity of the catecholamines were calculated, as follows: for noradrenalin 1.13·10⁴ M^?1, adrenalin 1.04·10⁴ M^?1, dopamine 7.6·10³ M^?1, and dopa 5·10³ M^?1. The antioxidative action of the catecholamines was linked with the presence of a free phenol group in their molecule. The mechanism of inhibition consists of interaction between catecholamines and free radicals leading the oxidation chains. It is postulated that the antioxidative action of catecholamines may have an important role in the regulation of the permeability of biological membranes.     

Effect of butylated hydroxytoluene on the life span of Drosophila bipectinata

The median and the maximum life spans of Drosophila bipectinata increased after feeding with various concentrations (10 μM, 10² μM and 10³ μM) of butylated hydroxytoluene (BHT). Insects fed on the diet supplemented with 10³ μM BHT had decreased rate of lipid peroxidation (measured by thiobarbituric acid test) with respect to the controls. It is suggested that the antioxidant BHT, which scavenges free radicals, prolongs the life span of Drosophila.     

In vitro induction of sister chromatid exchanges and chromosomal aberrations in peripheral lymphocytes of the oyster toadfish and american eel

A series of experiments was conducted to characterize the proliferation of oyster toadfish lymphocytes in medium containing 5-bromodeoxyuridine (BrdUrd) and to determine the effectiveness of cytogenetic endpoints for assessing the genotoxic effects of in vitro exposure of toadfish and eel lymphocytes to known mammalian clastogens. Although the rate of proliferation of toadfish lymphocytes was low compared to that of mammalian lymphocytes, the effects of increasing BrdUrd concentrations were similar, in that proliferation exhibited a concentration-dependent inhibition for concentrations above 10 μM BrdUrd, and sister chromatid exchange (SCE) frequencies exhibited a concentration-dependent increase for concentrations above 100 μM BrdUrd. Mitomycin C (MMC) and ethylene dibromide (EDB) induced concentration-dependent increases in chromatid-type exchange and SCE frequencies with least effective concentrations (control SCE frequency divided by the slope of the least-squares line) for SCE induction by MMC (6.8 × 10^?9 M) and EDB (2.6 × 10^?4 M) that were comparable to or slightly lower than those that have been obtained with mammalian in vitro systems. In vitro exposure of toadfish lymphocytes to dimethoate (DIM) induced a concentration-dependent increase in SCE frequency with a least effective concentration of 2.8 × 10^?3 M that was much higher than that observed with mammalian in vitro systems. In vitro exposure of American eel lymphocytes to MMC also induced a concentration-dependent increase in the frequency of chromosomal aberrations and SCEs with a least effective concentration for SCE induction of 2.0 × 10^?9 M. These results indicate that cytogenetic endpoints can be effectively scored with cultured lymphocytes from these and perhaps other fish species with comparable karyotypes that contain an average of at least 0.07 pg DNA/chromosome.     

Regulation of ERK2 phosphorylation by histamine in splenocytes

Histamine is implicated in allergic disease and asthma and ERK1/2 is involved in allergic inflammation including Th2 differentiation and proliferation. This study was designed to study the effects of histamine on ERK1/2 phosphorylation in splenocytes. C57/BL6 splenocytes were treated with different concentrations of histamine (10^?4 to 10^?11 M). Histamine (10^?4 M) increased ERK2 phosphorylation. There was, however, no significant effect seen at other concentrations (10^?11 to 10^?6 M). Surprisingly, H1 receptor agonist β-histine (10^?5 M), H2 agonist amthamine (10^?5 M), H3 agonist methimepip (10^?6 M), and H4 agonist 4-methyl histamine (10^?6 M), all increased ERK2 phosphorylation. H1R antagonist pyrilamine (10^?6 M), H2R antagonist ranitidine (10^?5 M), H3/H4R antagonist thioperamide (10^?6 M), and H3R antagonist clobenpropit (10^?5 M) inhibited histamine-mediated ERK2 phosphorylation suggesting that all four histamine receptor subtypes played some role in this phosphorylation. Because tumor necrosis factor-α (TNF-α) causes phosphorylation of ERK1/2, we investigated whether histamine acted via secretion of TNF-α to affect ERK1/2 phosphorylation. As a consequence, TNF-α knockout mice were used and we found that there was inhibition of ERK1 and ERK2 phosphorylation by H2, H3, and H4 agonists. This was in contrast to the wild-type splenocytes where histamine augmented the phosphorylation of ERK2 via H2, H3, and H4 receptors. In TNF-α knockout mice histamine did not affect the phosphorylation of ERK2 via H1 receptors. The results suggested that histamine indirectly caused the ERK2 phosphorylation via its effects on the secretion of TNF-α and these effects were mediated via H1, H2, H3, and H4 receptors.     

Cyclic AMP metabolism in asthma: studies with leukocytes and lymphocytes

The effect of isoproterenol on cyclic AMP (cAMP) levels in lymphocytes and leukocytes from asthmatic and normal individuals has been studied. Lymphocyte cAMP levels rose in response to 10^?8 to 10^?2 M isoproterenol; the dose-response curve was biphasic with a maximum response at concentrations of 10^?6 to 10^?4 M. At all drug concentrations the response of cells from asthmatic individuals was less than the response of cells from normal control subjects. The difference between the two groups was not statistically significant, however. Similarly, basal cAMP levels were lower in the cells of asthmatic as compared with normal individuals, but again the difference was not significant. When these two parameters were combined and the results expressed as absolute cAMP levels after isoproterenol treatment, a significant difference was observed. Both the unstimulated. cAMP levels and the response to isoproterenol of leukocytes from normal and asthmatic subjects were influenced by the incubation medium. Use of a medium buffered with tris as compared with bicarbonate-phosphate resulted in lower basal cAMP levels and increased responsiveness to isoproterenol. Treatment of normal leukocytes with serum from asthmatic individuals did not alter their response to isoproterenol.     

Characterization and localization of ouabain receptors in Schistosoma mansoni

Saturable ouabain binding sites were detected in intact male schistosomes. The K_D for binding of ouabain to Schistosoma mansoni was 2.6 × 10^?7 M and to Schistosoma japonicum 2.9 × 10^?7 M. The binding of ouabain to the receptor demonstrated pharmacological specificity. Binding sites, obtained by differential centrifugation, were associated with fractions containing tegument (58%) and microsomal membranes (33%). Binding sites were concentrated in tegumental membranes, i.e., a 19-fold enrichment of receptors was found in membranes isolated from intact schistosomes exposed to Triton X-100. The antiparasitic drugs praziquantel (IC₅₀ = 9 × 10^?7 M) and Ro-11-3128 (IC₅₀ = 5 × 10^?6 M) inhibit binding of [³H]ouabain to intact parasites in a pharmacologic specific manner. Both praziquantel and Ro-11-3128 are without effect (IC₅₀ > 10^?4 M) on [³H]ouabain binding to homogenates of S. mansoni. These findings indicate that ouabain receptors are present in S. mansoni and that these receptors represent Na⁺-K⁺ pump sites. In addition, the characteristics and location of these receptors are consistent with previous observations on the physiological action of ouabain on the parasite.     

The effect of cations on the alkaline dissociation of tobacco mosaic virus

Tobacco mosaic virus (TMV) dissociated into free ribonucleic acid (RNA) and protein when placed in 0.02 M Tris [tris (hydroxymethyl) amino methane] buffer, pH 9.0, containing bentonite, for 24 hr at 2°. Sodium, potassium, cesium, magnesium, and calcium cations protected against complete dissociation by stopping the stripping process at certain sites along the nucleic acid. These sites were the same for all the ions, but cation concentrations required for equivalent results varied from 10^?6 to 10^?3M for divalent cations and from 10^?3 to 1.0 M for monovalent cations.Partially stripped virus (PSV) particles of several lengths were separated by sucrose density-gradient centrifugation and characterized by infectivity, UV light absorption, electron microscopy, and ribonuclease sensitivity. These tests confirmed that treatment of TMV with cations under alkaline conditions can result in the formation of several classes of PSV with intact, infectious nucleic acid.     

Structural requirements for recognition of vasopressin by antibody; Thermodynamic and kinetic characteristics of the interaction

The thermodynamic and kinetic properties of two conventional antivasopressin antisera were studied. Values for binding affinity were determined by equilibrium measurements to be Kd = 1.4 and 2.7 × 10^?12M. The kinetic parameters were independently determined. The association rate constants (kas) were calculated by a pseudo-first-order analysis of binding kinetics (ka = 3.1 and 1.7 × 10⁷M^?1 sec^?1). The dissociation rate constants (kds) were measured by dissociating antibody-labelled antigen complexes with large excess of unlabelled antigen (kd = 1.6 and 1.7 × 10^?5 sec^?1). A fairly close agreement was achieved between equilibrium and kinetic evaluation of the affinity.The heterogeneity cannot be assessed through equilibrium experiments because of the very low concentrations of reagents to be handled. However, kinetic studies strongly suggested that the molecular heterogeneity with respect to affinity of the antisera is restricted to a narrow range (5 × 10^?13M to 7 × 10^?12M).Despite their very similar physicochemical properties these two antisera exhibited different fine specificities: the study of cross-reactivities with various analogues of the original hapten showed that one antiserum—5—is clearly directed against the C-terminal moiety of the molecule. The antigenic determinant is a sequential one and composed of the last four aminoacids Cys-Pro-Arg-GlyNH₂, while the other antiserum is not so sensitive to modifications of the last residue GlyNH₂.