Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

A calcium-binding sialoglycoprotein associated with an apparent eggshell membrane of Globodera rostochiensis

Energy dispersive X-ray microanalysis has been used to study the binding of two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, to a calcium binding site on the eggshell. Eggs were treated with either buffered ruthenium red or buffer only, and the levels of ruthenium and calcium that could be detected from the surface of eggshells were measured before and after repeated etching in a plasma oven. Counts for ruthenium increased during etching to a maximum after 8–12 min, just before the eggshell was eroded from the juvenile. The calcium levels on eggshells binding ruthenium red were suppressed relative to those from buffer. Similar experiments with lanthanum showed a significant increase in this inhibitor after 10 min etching and an accompanying suppression of calcium levels. The nature of the binding site was probed by measuring ruthenium red binding after pretreatment with selected enzymes. A non-specific protease, pronase, reduced the subsequent binding of this inhibitor but the site was resistant to trypsin in the presence of calcium. Neuraminidase also had an effect suggesting that the protein is a sialoglycoprotein. Phospholipase A2 but not Phospholipase C influenced the subsequent binding of ruthenium red. Phospholipase A₂ caused a loss of hatching ability and the degradation of a phospholipid from the eggshell. Ruthenium red binding was also reduced by treatment of eggshells with detergents or sonication. Apparently a calcium-binding sialoglycoprotein forms part of the integral proteins of an eggshell membrane located on the inner surface of the eggshell.     

The predominant contribution of platelets to baseline and ascorbate-stimulated increments in cyclic GMP in human mononuclear leukocyte preparations.

In nine consecutive experiments with Ficoll-Hypaque-purified human mononuclear leukocytes containing 2.8 (range 1.1–4.3) platelets per leukocyte, 2–5 mM sodium ascorbate produced a 14-fold (range, 7- to 18-fold) rise in guanosine 3′: 5′-cyclic monophosphate (cyclic GMP) from baseline levels of 0.103 ± 0.056 pmol/10⁷ mononuclear leukocytes. In five experiments with mononuclear leukocytes prepared by the Ficoll-Hypaque method from human blood depleted of platelets by defibrination, 2–5 mM sodium ascorbate produced a twofold (range, one- to fourfold) rise in cyclic GMP from baseline levels of 0.030 ± 0.012 pmol/10⁷ mononuclear leukocytes. Thus, platelets contribute substantially to baseline and ascorbate-stimulated levels of cyclic GMP in standard Ficoll-Hypaque preparations of mononuclear leukocytes. The rise in cyclic GMP concentration in mononuclear leukocyte preparations elicited by ascorbate was independent of a calcium requirement, persisted for up to 3 hr in the presence of ascorbate, and was prevented by the introduction of nonsteroidal anti-inflammatory agents such as aspirin and indomethacin (ID₅₀ = 105 and 23.5 μM, respectively).     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

NICOTINE AND α-BUNGAROTOXIN BINDING TO AXONAL AND NON-NEURAL TISSUES1

Abstract— Nicotine binds to homogenates of lobster walking leg nerve (K_d= 1.1 ± 0.3 μm , B_max= 2.4 ± 0.5 nmol/g wet tissue), horseshoe crab leg nerve (K_d= 0.11 ± 0.06 μm , B_max= 1.3 ± 0.6nmol/g), and kidney from 18-month-old rats (K_d= 0.8 ± 0.2 μm , B_max= 23 ± 9 nmol/g). The pharmacological sensitivities of nicotine binding to lobster and horseshoe crab leg nerve homogenates are similar to that of the axonal cholinergic binding macromolecule (ACBM) (Denburg et al., 1972) of lobster leg. nerve membrane, while the binding to rat kidney is sensitive to α-bungarotoxin but not atropine or curare. There was no nicotine binding to rat heart or spleen, or to kidney from younger rats; little or no binding to blue crab nerve or to Torpedo electroplax motor nerve; and little binding (around 0.1 nmol/g) to rat liver. [³H]α-Bungarotoxin bound reversibly (0.17 nmol/g) to lobster leg nerve membrane The implications of these results for the distribution and function of the ACBM, and for the specificity of α-bungarotoxin, are discussed.     

Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S   aureus among 3 different bacteria with limit of detection of 10³ colony‐forming unit per milliliter. The apparent K _a was 1.39 μM⁻¹ ± 0.3 μM⁻¹. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K _a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K _a = 1.56 μM⁻¹ ± 0.69 μM⁻¹ at 25°C to K _a = 1.03 μM⁻¹ ± 0.9 μM⁻¹ at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S  aureus aptamer to its target were also 9.44 μM⁻¹ ± 0.38 μM⁻¹ at 50mM, 1.60 μM⁻¹ ± 0.11 μM⁻¹ at 137mM, and 3.28 μM⁻¹ ± 0.46 μM⁻¹ at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S  aureus .     

Solubilization of High-Affinity, Guanine Nucjeotide-Sensitive μ-Opioid Receptors from 7315c Cell Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [³H]-etorphine with high affinity (K_D= 0.304 ± 0.06 nM; B_max= 154 ± 33 fmol/mg of protein). Of the membrane-associated [³H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [³H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [³H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ-selective ligand [³H][D-Ala²,A/-Me-Phe⁴,Gly⁵-ol]enkephalin also bound with high affinity (K_D= 0.79 rM; B_max= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

Schistosoma mansoni: partial purification and properties of ornithine-delta-transaminase.

Ornithine-δ-transaminase (OTA) (EC 2.6.1.13) was isolated from Schistosoma mansoni and purified more than 16-fold. Treatment of the worm homogenate with 0.4% deoxycholate (DOC) in the presence of 0.8 M KC1 and 0.15 M NaCl at pH 8.3 resulted in solubilization of 85% of the enzyme. Sonication and high-speed centrifugation were unnecessary. The solubilization procedure and the subsequent purification steps required the presence of the coenzyme pyridoxal phosphate. The optimal pH for OTA was 8.5 and the optimal incubation temperature was 55 C. Michaelis-Menten constants (K_m) for ornithine and α-ketoglutarate were 1.53 mM and 2.07 mM, respectively, in enzyme preparations with a specific activity of 22–29 μmoles/hr/mg protein. The enzyme showed a high affinity for α-ketoglutarate but considerably less affinity for oxaloacetate and pyruvate. High concentrations of α-ketoglutarate and ornithine inhibited the OTA activity. Similarly inhibitory were the structurally related amino acids isoleucine and serine and also oxaloacetate. The K_m for α-ketoglutarate in the presence of oxaloacetate was 1.3 mM and the V_max was 8.38 μmoles/hr/mg protein.     

Thermodynamic characterization of ethidium bromide binding to a unique site on yeast tRNAphe.

A self-consistent thermodynamic characterization of the binding of ethidium to yeast phenylalanine-specific tRNA at 25°C, pH 7.0, in 11 nM MgCl₂, 375 nM NaCl, and 25 mM sodium phosphate has been obtained. Two ethidium molecules bind per tRNA under these conditions. The stronger site has a dissociation constant equal to 1.9 ± 0.5 μM and ΔH_dis°′ = 12 ± 1 Kcal/mol, and the weaker sites has a dissociation constant equal to 24 ± 9 μM and ΔH_dis°′ = 8.9 ± 1.5 Kcal/mol. The average calorimetric ΔH_dis°′ for the to sites 10.6 ± 0.4 kcal/mol. The thermodynamics of binding to the stranger sites are most probably the thermodynamics of interaction between A·U (6) and A·U (7), the unique site identified by Jones and Kearns. The binding is enthalpically driven and classical hydrophobic interactions do not appear to be important in the binding reaction.     

Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages

Rabbit alveolar macrophages internalize α-macroglobulin ¹²⁵I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · ¹²⁵I-T at 37°C than at 0°C. At 0°C cell-bound αM · ¹²⁵I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · ¹²⁵I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · ¹²⁵I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · ¹²⁵I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM¹²⁵I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization.     

Synthesis, α-glucosidase inhibition and molecular docking study of coumarin based derivatives

We have synthesized seventeen Coumarin based derivatives (1–17), characterized by ¹HNMR, ¹³CNMR and EI-MS and evaluated for α-glucosidase inhibitory potential. Among the series, all derivatives exhibited outstanding α-glucosidase inhibition with IC₅₀ values ranging between 1.10 ± 0.01 and 36.46 ± 0.70 μM when compared with the standard inhibitor acarbose having IC₅₀ value 39.45 ± 0.10 μM. The most potent derivative among the series is derivative 3 having IC₅₀ value 1.10 ± 0.01 μM, which are many folds better than the standard acarbose. The structure activity relationship (SAR) was mainly based upon by bring about difference of substituent’s on phenyl part. Molecular docking studies were carried out to understand the binding interaction of the most active compounds.     

Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

Calorimetric experiments of Mn2+-binding to α-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change ΔH° of the binding of Mn²⁺ to apo-bovine α-lactalbumin; ΔH° = −90 ± 4kJ·mol⁻¹. The binding constants, K_Mn²⁺, calculated from the calorimetric and circular dichroism titration curves, are (4.6±1) · 10⁵M⁻¹, respectively. Batch calorimetry confirms the competitive binding of Ca²⁺, Mn²⁺ and Na⁺ to the same site. The relatively small enthalpy change for Mn²⁺ binding compared to Ca²⁺ binding favours a model of a rigid and almost ideal Ca²⁺-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an α-helix which is directly connected to the complexating loop.     

Evidence for the involvement of calcium in the hatching of Globodera rostochiensis

Low concentrations of ruthenium red and lanthanum chloride inhibited hatching of juveniles from eggs in cysts of Globodera rostochiensis in potato root diffusate. Pro-bit analysis for 31 cysts at seven concentrations of ruthenium red showed that 50% inhibition with 95% fiducial limits occured at 47 ± 23 μm; a similar value of 59 ± 14 μm was obtained using eggs removed from cysts. Results for 10 to 20 cysts at six concentrations of lanthanum chloride suggested a somewhat higher value for 50% inhibition of 110 ± 83 μM. In contrast hatching of eggs in cysts of Heterodera schachtii in water was unaffected by 5 ITIM lanthanum chloride and 625 μM ruthenium red, concentrations which cause over 90% inhibition of hatch in G. rostochiensis.
Two calcium ionophores synergised hatching of a 1971 population of G. rostochiensis in dilute diffusate. Optimal concentrations of 2 μM for A23187 and 10 μM for BrX537A increased the hatch from 17 ± 3–6 juveniles/cyst to 114 ± 44 juveniles/cyst and 138 ± 40 juveniles/cyst respectively. Ionophores in the absence of diffusate hatched very few eggs of this population but caused a greater hatch in a second (1975) population which gave a high hatch in water of 43 ± 10 juveniles/cyst. This was increased by A23187 to 181 ± 41 juveniles/cyst. The results with both the inhibitors and the ionophores suggest that hatching in G. rostochiensis might be a calcium-mediated process.     

Differential Inhibition by α-Conotoxin-MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α-conotoxin-MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)-anatoxin-a-evoked release of [³H]dopamine from rat striatal synaptosomes and slices. α-Conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC₅₀ = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα-conotoxin-MII. On perfused striatal synaptosomes and slices, α-conotoxin-MII dose-dependently inhibited [³H]dopamine release evoked by 1 µM (±)-anatoxin-a with IC₅₀ values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α-conotoxin-MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)-anatoxin-a-evoked [³H]dopamine release by α-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α-conotoxin-MII-insensitive nAChR.     

Comparative characteristics of binding kinetics of specific antagonists by α<Subscript>1</Subscript>- and α<Subscript>2</Subscript>-adrenoceptors in rat cerebral cortex membranes

The binding of specific nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosine and [³H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α₁-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were: K _d= 1.56 ± 0.17 nM, B _max = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.94 ± 0.08 nM, B _max = 12.77 ± 3.17 fmol/mg protein, n = 2. For α₂ -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α₁ - and α₂-adrenoceptor antagonists the dissociation constants (K _d) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α₂-adrenoceptors is two times lower than that of α₁-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = B _max/2K _d) of the ligand binding to α₁-adrenoceptors is 2.3 times higher than that to α₂-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α₁- and α₂ -adrenoceptors in rat cerebral cortex exist as dimers.     

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average V_max of 0.86±0.01 μmol min^–1mL^–1 and K_M of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (K_i=64.0±0.53 nM), competitively inhibited by bacitracin (K_i=0.16±0.01 mM) and bestatin (K_i=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC₅₀ value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn²⁺ and Ca²⁺, for which a mixed inhibition mechanism was observed (K_i values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca²⁺ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn²⁺ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.