Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and thermolysin

Neutral salts activate and stabilize thermolysin. In this study, to explore the mechanism, we analyzed the interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and thermolysin by ANS fluorescence. At pH 7.5, the fluorescence of ANS increased and blue-shifted with increasing concentrations (0–2.0?μM) of thermolysin, indicating that the anilinonaphthalene group of ANS binds with thermolysin through hydrophobic interaction. ANS did not alter thermolysin activity. The dissociation constants (K_d) of the complex between ANS and thermolysin was 33?±?2?μM at 0?M NaCl at pH 7.5, decreased with increasing NaCl concentrations, and reached 9?±?3?μM at 4?M NaCl. The K_d values were not varied (31?34?μM) in a pH range of 5.5?8.5. This suggests that at high NaCl concentrations, Na⁺ and/or Cl^– ions bind with thermolysin and affect the binding of ANS with thermolysin. Our results also suggest that the activation and stabilization of thermolysin by NaCl are partially brought about by the binding of Na⁺ and/or Cl^– ions with thermolysin.     

A dopamine- and octopamine-sensitive adenylate cyclase in the nervous system of Octopus vulgaris

• 1.1. Adenylate cyclase activity was assayed in the optic lobe of Octopus vulgaris.
• 2.2. Both octopamine and dopamine stimulate the octopus adenylate cyclase, apparently by competing with the same receptor site.
• 3.3. (±)-2-Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene-HBr (6,7-ADTN) and a number of phenylethanolamine derivatives stimulate the octopus adenylate cyclase activity.
• 4.4. The dopamine D-1 antagonists R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SCH-23390) and (±)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SKF-83566) are unable to antagonize the effects of dopamine and octopamine, and similarly ineffective is the agonist (±)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol-HCl (SKF-38393).
• 5.5. No detectable binding of labelled SCH-23390 occurs on membrane preparations from octopus optic lobe.

     

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry

Cannabinoid CB₁ receptors are highly expressed in the striatum where they are known to be co‐localized with dopamine D₂ receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D₂ receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration‐dependent manner (IC₅₀: 3.25 × 10^?8 M). The CB₁ receptor agonist WIN55212‐2 (WIN; 1 μM) had no effect on single pulse dopamine release (93.9 ± 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 ± 4.2% vs. quinpirole + WIN, 48.2 ± 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB₁ receptor anatgonist [N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] (AM‐251, 1 μM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 ± 7.9% control (peak 4 to 1 ratio measurement) and 66.9 ± 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse‐evoked dopamine release (4P1Hz: quinpirole, 28.4 ± 4.8% vs. WIN + quinpirole, 52.3 ± 1.2%; 10P5Hz: quinpirole, 29.5 ± 1.3% vs. WIN + quinpirole, 59.4 ±7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM‐251 (1 μM). This is the first report demonstrating a functional, antagonistic interaction between CB₁ receptors and D₂ autoreceptors in regulating rat striatal dopamine release.     

Salivary progesterone for the assessment of the ovarian function in the capuchin monkey (Cebus apella)

Salivary and plasma progesterone were measured in normally cycling (n=10) and castrated (n=4) femaleCebus monkeys (Cebus apella). During the follicular phase, progesterone levels in saliva ranged between 0.05 and 1.40 ng/ml and in the luteal phase they increased to between 0.22 and 4.70 ng/ml. These values represented on average 6.5 and 3.2% of those values measured in plasma, for the follicular and luteal phases, respectively. The regression analysis of the steroid concentrations in both fluids showed a highly significant correlation (r=0.8985,n=180,P<0.0001). Ovariectomized monkeys had consistently low salivary (0.37±0.02 ng/ml) and plasma (4.70±0.25 ng/ml) progesterone, showing a low, but significnat, correlation coefficient (r=0.2592,n=58,P=0.047). The ratio of plasma/salivary progesterone was significantly higher in the luteal phase (31.09±1.65) than in the follicular phase (23.06±2.26) and in castrated monkeys (16.00±1.38). The free fraction of progesterone constituted 5.3±0.2% of the total plasma progesterone during the follicular phase and 3.3±0.1% during the luteal phase. Ovariectomized monkeys showed a significantly higher percentage of free progesterone in plasma (7.7±0.1%). In contrast, free progesterone made up 64.4 and 70.9% of the total salivary progesterone for the follicular and luteal phases, respectively. The proportion of free progesterone in castrated animals was within the range observed in cycling animals. We suggest that the levels of progesterone in the saliva of capuchin monkey follow a pattern similar to that for plasma progesterone, reflecting the free steroid fraction. Thus, the measurement of such steroid in saliva may offer a valuable alternative to plasma determinations for the assessment of the ovarian function inCebus and probably other New World monkey species.     

Testosterone binding in the chick oviduct

A novel androgen receptor was observed in estrogen-stimulated chick oviducts but not in unstimulated oviducts. This binding component showed a preference for androgens and could therefore be distinguished from oviduct receptors for estadiol and progesterone. Testosterone was tightly bound having a dissociation constant (Kd) of 2.7 × 10^?10 M. Sucrose gradient centrifugation, under low ionic strength conditions, showed testosterone to be bound as an 85 complex. These binding properties, plus the estrogen dependency of this component, suggest its role as a biological receptor for androgens.     

Revealing binding interaction between seven drugs and immobilized β2‐adrenoceptor by high‐performance affinity chromatography using frontal analysis

The development of new approaches to study the affinity between ligands and G‐protein‐coupled receptors proves to be of growing interest for pharmacologists, chemists, and biologists. The aim of this work was to determine the binding of seven drugs to β₂‐adrenoceptors by frontal analysis using immobilized receptor stationary phase. The dissociation constants (K_d) were determined to be (3.16 ± 0.09) × 10^?4 M for salbutamol, (4.29 ± 0.12) × 10^?4 M for terbutaline, (6.19 ± 0.16) × 10^?4 M for methoxyphenamine, (2.11 ± 0.07) × 10^?4 M for tulobuterol, (1.82 ± 0.11) × 10^?4 M for fenoterol, (9.75 ± 0.24) × 10^?6 M formoterol, and (9.84 ± 0.26) × 10^?5 M for clenbuterol. These results showed a good correlation with the data determined by radioligand binding assay. Further investigations revealed that the dissociation constant mainly attributed to the number of hydrogen bonds in the structures of ligands. This study indicates that affinity chromatography using immobilized receptor stationary phase can be used for the direct determination of drug‐receptor binding interactions and has the potential to become a reliable alternative for quantitative studies of ligand–receptor interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Receptor and Biological Response to Estriol in the Fetal Uterus of Guinea Pig

Abstract

The binding of ³H-estriol was examined in the fetal uterus of guinea pig. The physico-chemical characteristics of the binding of ³H-estriol to macromolecules are similar to the typical receptor protein for estrogens. Different estrogens (estriol, estradiol, estrone and diethylstilbestrol) compete with this binding but progesterone and testosterone have no effect. The binding affinity has a Kd of 5.5 ± 1.6 ± 10^?10M. By ultra-centrifugation in sucrose gradient, two specific components with sedimentation coefficients of 8 and 45 are found. Competition studies suggest that the same specific binding sites may be present for estriol (E₃) and for estradiol. The s.c. administration of E₃ to the pregnant guinea pig (1 mg/day per kg body weight for 3 days) provokes two biological responses in the fetal uterus: a uterotrophic effect and a significant increase in the progesterone receptor. The increase in the fetal uterine weight is 50–70% in relation to the non-treated animals and the progesterone receptor concentration is 10–14 times higher than in the control animals. These effects are similar (or slightly higher) than in animals primed with equimolecular quantities of estradiol. In contrast, single daily injections of E₃ to newborn guinea pig, results only in weak uterotrophic activity.

It is concluded that estriol is capable of causing a biological response in the uterus during intra-uterine life.     

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei,and chromatin from rat hepatocytes

Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth-modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (K_s1 = 2.4 ± 1.6 μM; K_s2 = 90 ± 17 μM) that corresponded to microsomal binding sites (K_d1 = 1.0 ± 0.9 μM; K_d2 = 57 ± 13 μM) resolved by ³H-histamine binding; additional low affinity (K_d3 ∼ 3 mM), and probably physiologically irrelevant, sites were resolved only by ³H-histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by ³H-histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for ³H-histamine binding: in microsomes, K_i = 9.8 ± 5.8 μM; in nuclei, K_i = 13.7 ± 3.1 μM; in chromatin, K_i = 46 ± 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine > spermidine ≫ putrescine. In contrast, histamine did not compete for ³H- spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth-regulatory endogenous bioamines and, as shown previously, of exogenous growth-modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines. J. Cell. Biochem. 69:233–243, 1998. © 1998 Wiley-Liss, Inc.     

Erythrocyte agglutination by wheat germ agglutinin: ionic strength dependence of the contact seam topology

Abstract

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K_D of 138 ± 55 nM, a ΔH of ?9708 ± 1195 cal/mol and a ΔS of ?1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K_D of 3.2 ± 0.8 μM, a ΔH of ?2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.     

A estradiol-17beta receptor in the reproductive system of the female of Octopus vulgaris: characterization and immunolocalization

In this study, for the first time we have identified an estradiol-17beta receptor (ER) in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present in the cytosol, but not in the nuclear extract of the ovary and the oviduct. A steroid specificity competition assay showed that 3H-estradiol-17beta binding activity showed a preference for estradiol-17beta. DNA-cellulose chromatography confirmed the presence of one 3H-estradiol-17beta binding component. By using antibodies anti ER (578-595), we have localized by Western blotting one band of about 70 kDa. ER immunoreactivity has been localized in the nuclei of the follicle cells of the ovary, in the nuclei of the epithelium lining the proximal portion of the oviduct and in the nuclei, and in the cytoplasm of the inner region of the oviducal gland and in the cytoplasm of the outer region of the oviducal gland. These data, taken together, provide evidence that in Octopus vulgaris the ER has biochemical and immunohistochemical characteristics resembling those of ER in vertebrates.     

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.     

New Drugs for the Na+/H+ Exchanger. Influence of Na+ Concentration and Determination of Inhibition Constants with a Microphysiometer

The NHE-1 isoform of the Na⁺/H⁺ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na⁺/H⁺ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na⁺/H⁺ antiporter. The Na⁺/H⁺ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K _M = 30 ± 4 mm for Na⁺. Addition of either one of the four inhibitors decreased the acidification rate. The IC₅₀ values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC₅₀ values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na⁺/H⁺ antiporter and allows a quantitative kinetic analysis of the proton excretion rate. Received: 3 September 1998/Revised: 20 November 1998     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

Evidence for acetylcholine as a neurotransmitter in the statocyst of Octopus vulgaris

Summary Evidence is presented for acetylcholine as neurotransmitter in the sensory epithelia (macula and crista) of the statocyst of Octopus vulgaris, based on the following techniques: (i) histochemical assay of acetylcholinesterase at light- and electron-microscopical levels, in combination with the detailed knowledge of the ultrastructural and neuronal organization of the receptor epithelia; (ii) lesion/degeneration experiments of the efferent fibre system; (iii) radiochemical assay of acetylcholine; and (iv) bioassay of acetylcholine. All data support the hypothesis that in the statocyst of O. vulgaris acetylcholine acts as a neurotransmitter in the efferent fibre system.This paper is dedicated to Professor Franz Huber, Seewiesen, on the occasion of his 60th birthday     

Characterization of a distinct glucocorticoid-binding protein in the lactating mammary gland of the rat

Specific substances binding [³H]triamcinolane acetonide were detected in the cytosol fraction of the lactating mammary gland of the rat using sucrose gradient centrifugation. These receptors, which were protein in nature, exhibited sedimentation coefficients of 7–8 S and dissociated into lower molecular weight components sedimenting at 4–5 S when separated on sucrose gradients containing 0.4 M KCl. The cytoplasmic form of the binding protein was relatively specific for glucocorticoids although progesterone inhibited binding significantly. The dissociation constant (K_d) of the receptor-ligand complex was in the range of 10⁻⁸ M. p-Chloromercuribenzoate diminished the ligand-binding capacity of the receptor suggesting a role for sulfhydryl groups in the binding reaction. Cytosols from mammary tissue obtained from virgin and pregnant rats revealed a paucity of binding sites as compared to those in the lactating gland. Examination of ligand-binding specificity indicates that these glucocorticoid-binding sites are distinct and easily discriminated from those of either the estrogen receptor of the mammary gland or the triamcinolone-binding component in plasma.     

Immunohistochemical Study of Leucine-Enkephalin and Delta Opioid Receptor in Mantles and Feet of the Octopus Octopus ocellatus Gray

The study was supposed to determine whether leucine-enkephalin (leu-Enk) or delta opioid receptor was presented in mantles and feet of the Octopus Octopus ocellatus. The results showed that they were found in the mantles, wrists, interbrachial membrane, and funnels of the Octopus Octopus ocellatus. The immunoreactivities of leu-Enk and delta opioid receptor were observed in the epithelial cells of dorsal mantle, wrist and interbrachial membrane. Strong one was presented in both the connective tissue which was close to the epithelium of dorsal mantle, wrist and interbrachial membrane and a small amount of connective tissue cells and nerve fibers, while less positive in other parts. A little of immunoreactive material was also observed in the ventral mantle and funnel, for example, weak one was in the epithelial tissue, the adjacent cells and nerve fibers. The different densities of leu-Enk and delta opioid receptor in mantles and feet of the Octopus O. ocellatus may be related to the different functions.     

Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [³H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 10⁸ M⁻¹ at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [³H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [³H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [³H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [³H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C . Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [³H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K⁻¹ · mol⁻¹.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Countercurrent distribution and partition of Chlorella-sensitive and -resistant strain to the lytic agent in two-phase system. I

The influence of various buffering ions and NaCl concentrations on the distribution of Chlorella cells of two strains having differing surface and size were studied in an aqueous dextran-polyethylene glycol phase system using the one-step partition and CCD method.As models for these experiments we used an artificial mixture of Chlorella pyrenoidosa A 24 or Chlorella sp. 366 (strains sensitive to the infective lytic agent) and cells of Chlorella vulgaris B. resistant to this agent. The average size of Chlorella pyrenoidosa A 24 cells was 5.3 ± 0.53 μm and of Chlorella vulgaris B. 6.8 ± 0.55 μm.The condition of complete distribution of this algal mixture in 0.01 M sodium and potassium phosphate buffer is fulfilled when the concentration of NaCl is equal to or greater than a critical concentration of 15 mM of NaCl in the first, and 10 mM of NaCl in the second buffer.In buffers containing—COOH groups at pH 7.3 and low molarity a complete separation of mixture components was obtained. The addition of NaCl to maleate- and fumarate-NaOH buffers of pH 7.3 slightly worsened the separation effect which was visualized when decreasing the r_max difference value of both algal components on comparison with the separation obtained in a system without NaCl.At pH 6.0 in various buffers containing —COOH groups a complete separation of the algal components of the mixture was observed only when NaCl was absent. The addition of NaCl to some buffers of pH 6.0, e.g. maleate, malonate, and succinate, destroyed completely the successful separation and gave only a common peak of small r_max value.The fumarate buffer of pH 6.0, in the absence of NaCl, was an exception and gave only one common peak of small r_max value in contrast to its stereo-isomer maleic acid where two peak separation was obtained.     

The daily activity pattern of Octopus vulgar is in a natural habitat

The homes of Octopus vulgaris in shallow Mediterranean water were found and marked by divers using SCUBA; hourly inspection by day and night was maintained for eleven days. It was shown’ that O. vulgaris in this habitat generally hunts throughout the night, with only short excursions from the hole by day.