Rat Frontal Cortex β1-Adrenoceptors Are Activated by the β3-Adrenoceptor Agonists SR 58611A and SR 58878A but Not by BRL 37344 or ICI 215,001

Abstract: SR 58611A, a selective agonist of gut and brown adipose tissue β₃-adrenoceptors (β₃ARs), has been reported to have antidepressant-like activity in rodents by indicating brain β₃ARs as the sites of this property. SR 58611A and its acid metabolite SR 58878A, as opposed to BRL 37344, ICI 215,001, and CGP 12177, increased cyclic AMP levels in rat frontal cortex. ICI 215,001, differently from BRL 37344, at concentrations in the millimolar range antagonized norepinephrine- or (−)-isoproterenol-stimulated adenylyl cyclase partially. The increase of cyclic AMP levels induced by SR 58878A was blocked selectively by β₁AR antagonist CGP 20712A but not by β₂AR antagonist ICI 118,551. In addition, PCR analysis did not reveal β₃AR mRNA, and no specific β₃AR binding sites were detected by [³H]CGP 12177 in rat frontal cortex. When down-regulation of the β₁AR ligand binding and mRNA levels had been induced in frontal cortex by chronic administration of imipramine, SR 58878A as well as norepinephrine and (−)-isoproterenol increased the cyclic AMP production less markedly. Our findings indicate that β₃ARs are absent in the adult rat frontal cortex, and that various β₃AR agonists differently affect the frontal cortex β₁ARs, indicating that SR 58611A may exert its putative antidepressant effect acting on the frontal cortex β₁ARs.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

A comparative study of blood oxygen transport in turbot and sea bass: effect of chronic hypoxia

A comparative study of blood oxygen binding and carrying capacities of turbot Scophthalmus maximus and sea bass Dicentrarchus labrax , two fish species differing in their demand for oxygen, was carried out under three levels of chronic hypoxia ( P o ₂ = 93, 65 and 40 mmHg) for 40 days. Blood O₂ affinity in normoxia was moderately high in both species ( P ₅₀ was c . 12–13 mmHg at pH 7·7). The Bohr factor was significantly lower in turbot (−0·52) than in sea bass (−0·85). In both species, blood O₂ affinity was not significantly affected by oxygen depletion whatever its level and duration. In turbot, however, P ₅₀ appeared to slightly decrease at the two more severe levels of hypoxia. In both species, blood O₂ carrying capacity was not affected by hypoxia and remained twice as high in sea bass than in turbot.     

SELECTIVE PERMEABILITY OF THE EXTRACELLULAR ENVELOPE OF THE MICROALGA SPONDYLOSIUM PANDURIFORME (CHLOROPHYCEAE) AS REVEALED BY ELECTRON PARAMAGNETIC RESONANCE

The aim of this work was to investigate the role of the polysaccharide sheath of the microalga Spondylosium panduriforme (Chlorophyceae, Desmidiaceae) in the selective permeability and transport of molecules into the interior of the cell. We have used the electron paramagnetic resonance (EPR) technique applied to a variety of spin labels of a hydrophobic nature with different substitutents on the ring (−OH, =O, −N=C=S, −NH₃⁺, and others). The spin label EPR signals were destroyed as a consequence of metabolic processes once the spin probes had entered the cells. The decay time of the EPR signal was regulated by the diffusion mechanism across the polysaccharide sheath, cell wall, and membrane. To discriminate the effect of the polysaccharide sheath from that of the cell wall and membrane, the polysaccharide sheath was removed by ultrasonic treatment. The decay times for the cells without capsule were faster than those for intact cells, and a possible mechanism of interaction involving hydrogen bonds between the spin labels and the −OH groups of the polysaccharide sheath is presented. These were expressed by their diffusion and friction coefficients as derived from Ficks' Second Law and the Einstein-Stokes equation and were summarized in terms of diffusion coefficients ( D ₁) for the capsule medium in the order: =O < −OH < −phe < −H < −N=C=S; and for cell wall and membrane ( D ₂): −OH < −H < =O < −NH₃⁺≅−phe < −N=C=S. For the friction coefficients ( f ₁ and f ₂), the order was inverted. These results suggest the capsule plays a role in selectivity as a result of polar interactions with the spin labels.     

Multiple Laminin Binding Proteins in Human Fetal Heart

The possibility of occurrence of laminin binding proteins in cardiac tissue under different stages of growth was examined by affinity chromatography of the soluble fraction of human fetal myocardial plasma membrane over Ln-Sepharose. A 67 kDa protein was isolated by elution with glycine/HCl buffer containing 1 M NaCl and visualized as a coomassie stainable band on SDS gel electrophoresis under reducing conditions. Dot blot assays of the radioiodinated protein revealed the binding of 67 kDa protein with high affinity to laminin in a cation independent manner. This protein appears to be present in relatively higher amounts in tissues from early stage fetus. The occurrence of cation dependent laminin binding proteins was also examined by affinity chromatography. Electrophoresis of the EDTA eluate under reducing conditions followed by silver staining showed two prominent bands with average molecular size 130 and 174 kDa which under non-reducing conditions appeared as two bands with average molecular weight of 115 and 135 kDa. Using radioiodinated protein in dot blot assays, its binding to Ln was found to be maximum in the presence of Mn⁺⁺ ions. Immunoblotting using anti-β₁ integrin antibodies showed that 115 kDa protein is a β₁ integrin suggesting the possibility of this protein belonging to the integrin group of receptors. The occurrence of multiple laminin binding proteins and the relative abundance of one of these proteins viz. the 67 kDa protein during early stages than in late stage tussue suggest a possible role for these proteins in cellular interactions with laminin during myocardial tissue development.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Borrelia burgdorferi BBB07 interaction with integrin alpha3beta1 stimulates production of pro-inflammatory mediators in primary human chondrocytes

Borrelia burgdorferi , the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin α₃β₁ on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin α₃β₁ and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg–Gly–Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin α₃β₁ and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin α₃β₁. This interaction is specific, as P66, another integrin ligand of B. burgdorferi , does not activate signalling through α₃β₁. In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin α₃β₁ and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.     

Cloning and Characterization of a Mouse σ1 Receptor

Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ₁ cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ₁ receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[³H]pentazocine binding with a selectivity profile consistent with σ₁ receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[³H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[³H]pentazocine binding site with characteristics of a murine σ₁ receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.     

Developmental Changes of Cerebral Cortical [3H]Clonidine Binding in Rats: Influences of Guanine Nucleotide and Cations

Abstract: Cerebral cortical [³H]clonidine binding and influences of GTP and cations were investigated in developing rats. The results from Scatchard plots were compatible with the presence of two populations of binding sites [high-affinity binding ( K_D = 0.59 n M ) and low-affinity binding ( K_D = 7.12 n M )] in 70-day-old rats but only high-affinity binding ( K_D = 0.27 n M ) on day 1. Low-affinity binding was detectable on day 7. K_D values in high- and low-affinity binding were not significantly changed during development after 7 days. B_max of high-affinity binding reached a peak on day 15, and the value of low-affinity binding gradually increased with age. The addition of 10 μ M GTP caused a significant reduction in B_max of high-affinity binding after day 7. Neither K _D nor B_max of low-affinity binding was affected by 10 μ M GTP during development. NaCl (10 and 100 m M ) diminished the binding on days 7 and 70. MnCl₂ (0.1 and 1.0 m M ) markedly increased the binding on days 15 and 70 but not on day 7. It is suggested that: (1) single binding sites of α₂-adrenoceptors with higher affinity seem to be present on day 1; (2) low-affinity binding appears on day 7; (3) the number of high-affinity binding sites reaches a peak on day 15, followed by changes in populations of high-affinity as well as low-affinity sites without changing affinity; (4) the regulatory mechanism in α₂-receptors by guanine nucleotide reaches functional maturity between days 1 and 7; and (5) the involvement of Na⁺ and Mn²⁺ in α₂-receptor binding becomes functional by days 7 and 15, respectively.     

Physical factors affecting the toxicity of sprays to stored product insects; the quantity of carrier in which a given amount of active ingredient is applied

The problem of whether a given amount of active ingredient is more effectively applied in concentrated or dilute form is discussed. If y is percentage mortality and x ₁ and x ₂ are respectively log-concentration of active ingredient and log-volume (or deposit) of insecticide applied, y may be regarded as a uniform, continuous function of x ₁ and x ₂. The amount of active ingredient is constant, i.e. x ₁+ x ₂= k , so that
dx/dx₁=−dy/dx₂=δy/δx₁−δy/δx₂
Probit mortality, Y , can be substituted for y in (4). Thus, whether an active ingredient is better applied in concentrated or dilute form depends on the relative magnitudes of ∂ y /∂ x ₁ and ∂ y /∂ x ₂, or of ∂ Y /∂ x ₁ and ∂ Y /∂ x ₂. Equation (4) is true whenever an insecticide consists of an active ingredient in a diluent, whatever the dosage-mortality relationship. Previous work is discussed in the light of (4) and its probit form, and it appears that the concentration at which an active ingredient is best applied can depend upon the nature and quantity of the active ingredient, and the method of application of the spray. There may be other facto*** The probit form of (4) is applied to the probit plane and confirmed experimentally. Flour beetles, Tribolium castaneum Herbst were sprayed with pyrethrins in Shell oil P 31, and it was found that ∂ Y /∂ x ₁ > ∂ Y /∂ x ₂, so that a given quantity of pyrethrins was more toxic in concentrated solution than in dilute.     

Effects of sulfate and nitrate on K+ uptake and growth of wheat and cucumber

The uptake of K⁺ ion was studied in the roots of wheat ( Triuicum aestivum L. cv. GK Szeged) and cucumber ( Cucumis sativus L. cv. Budai csemege) seedlings grown in nutrient solution under nitrogen and sulfate stress conditions. Seedlings pretreated with 1 or 10 m M NaNO₃, absorbed more K⁺ than those treated with 0.1 m M NaNO₃. However, the posteffect of NaNO₃ was considerably influenced by the Na₂SO₄, treatment. The results suggest that, at least partly, a feed-back regulation of K⁺ uptake may occur. However, due to the high Na⁺ contents of the roots, a Na⁺ effect in this process cannot be excluded. The growth and dry matter yields of the roots and shoots were strongly influenced by the SO²⁻/₄ and NO⁻/₃ supply of the plants. Appreciable differences were experienced between wheat and cucumber seedlings. The optimum SO²⁻/₄ concentration of the growth solution for maximal growth varied considerably between the species, and was also different for the roots and the shoots in a given species.     

Phospholipase Cγ1 in Bovine Rod Outer Segments: Immunolocalization and Light-Dependent Binding to Membranes

Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ₁, γ₁, and δ₁. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ₁ was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ₁ showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ₁ on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca²⁺]_free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ₁ in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.     

Growth and reproduction of Mesopotamian spiny eel (Mastacembelus mastacembelus Banks & Solander, 1794) in Ataturk Dam Lake (Şanliurfa), Turkey

Age at length, growth and reproduction of 220 Mastacembelus mastacembelus specimens from the Atatürk Dam Lake were studied from July 2005 to July 2006. Total lengths and weights ranged from 7.0 to 85.0 cm and from 6 to 1100 g, respectively. Maximum age was 13 years. The regression model fitted to length and weight data was W  =   0.0228  L ^2.43 for males and W  =   0.0029  L ^2.95 for females. The von Bertalaffy growth equations for males and females were L _t  = 99.2 [1−e^{−0.11 ( t −0.12)}] and L _t  = 69.2 [1−e^{−0.26 ( t −0.35)}], respectively. Males dominated especially at an older age; the overall sex ratio was 1 : 0.63 (M:F). The breeding period was from May to July. Fecundity ranged from 2540 to 24 000 eggs per female.     

Inheritance of Anthracnose Resistance in the Common Bean Differential Cultivar G 2333 and Identification of a New Molecular Marker Linked to the Co-42 gene

The inheritance of anthracnose resistance of the common bean ( Phaseolus vulgaris L.) differential cultivar G 2333 to Colletotrichum lindemuthianum races 73 and 89 was studied in crosses with the susceptible cultivar Rudá. The segregation ratios of 15 : 1 in the F₂ and 3 : 1 in the backcrosses to Rudá indicate that for each of the races tested there are two independent resistance loci in G 2333. A random amplified polymorphic DNA (RAPD) molecular marker (OPH18_1200C) linked in resistance to race 73 was identified in a BC₃F_2:3 population derived from crosses between Rudá and G 2333. A RAPD molecular marker OPAS13_950C, previously identified as linked to gene Co-4² , was also amplified in this population. Co-segregation analyses showed that these two markers are located at 5.6 (OPH18_1200C) and 11.2 (OPAS13_950C) cM of the Co-4² gene. These markers were not present in BC₁F_2:3 plants resistant to race 89 indicating that this population carries a different resistance gene. DNA amplification of BC₁F_2:3 plants with RAPD molecular marker OPAB_450C, previously identified as linked to gene Co-5 , indicated that this gene is present in this population.     

Rod visual pigment changes in the elver of the eel Anguilla anguilla L. measured by microspectrophotometry

The visual pigments in the retinal rods of elvers ( Anguilla anguilla L.) were examined by microspectrophotometry and high performance liquid chromatography. The glass elver stage had a mixture of rhodopsin P501₁ and porphyropsin P523₂ with the former predominating (P501₁:P523₂= c. 60:40). More mature pigmented elvers had a predominance of the porphyropsin P523₂ (P501₁:P523₂= c. 10:90). The shift between the proportions of each pigment was monitored over a 7-month period and was shown to occur in individual retinal rods. This shift is a reverse of the pigment shift that occurs in adult eels during their downstream migration.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.