Influence of Carbicron (O-[(2-butenoic acid)-N,N-dimethylamide-3-YL] O,O-dimethylphosphate) on some biochemical and biophysical parameters of rat liver membranes

• 1.1. Treatment of rats with carbicron induced a reduction of the phospholipids in both microsomal and plasma membranes.
• 2.2. A decrease of the structural order parameter (S_DPH) and an increase of the pyrene excimer-to-monomer fluorescence ratio (I_E/I_M) was also observed, indicating membrane fluidization.
• 3.3. The specific activity of membrane-bound phospholipase A₂ and phospholipase C were decreased in both types of membranes, whereas acyl-CoA:lysophosphatidylcholine acyltransferase activity was augmented due to carbicron treatment.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
• 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
• 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
• 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower M_r, Stokes-radius and diffusion coefficient.
• 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the M_r of the subunits were unaffected.
• 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
• 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
• 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.

     

Intestinal l-methionine transport in the cultured gilthead bream (Sparus aurata)

• 1.1. The influx and transepithelial movements of l-methionine and its effects on the electrophysiology and Na-Cl-transport in upper and lower intestine of the cultured fish, Spanis aurata, were measured.
• 2.2. The K_m and V_max of l-methionine influx into the tissues were higher in lower intestine than in upper intestine. A prominent diffusion-like transport component was also measured in both segments during influx experiments.
• 3.3. Net transepithelial fluxes of l-methionine (1 mM) were observed in both upper and lower intestine, this transport being Na⁺-dependent.
• 4.4. The two intestinal segments exhibited an electrical potential difference (PD) and a short circuit current (I_sc) serosa negative or near zero. Tissue conductance (G_t) was higher in posterior than in lower intestine.
• 5.5. Addition of l-methionine to the mucosal side of lower or upper intestine did not induce changes in PD in either part.
• 6.6. Isotopic fluxes of Cl⁻ or Na⁺ measurements under short circuit conditions showed that there were no net Cl⁻ or Na⁺ transport in either part.
• 7.7. l-Methionine additions to the mucosa did not induce changes in unidirectional fluxes of Cl⁻ or Na⁺ or in the (I_sc) in either the anterior or posterior intestine.

     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. I. Effects of fructose-2,6-bisphosphate and divalent cations

• 1.1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg²⁺ and Mn²⁺.
• 2.2. Mg²⁺ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory.
• 3.3. Mn²⁺ produces a 10-fold rise in V_max higher than Mg²⁺. [Mn²⁺]_0.5 is much larger than [Mg²⁺]_0.5. At elevated [Mn²⁺] inhibition is observed.
• 4.4. Mg²⁺ and Mn²⁺ produce antagonistic effects on the inhibition of the enzyme by high substrate.
• 5.5. Fru-2,6-P₂ inhibits the enzyme by rising the S_0.5 and favouring a sigmoidal kinetics.
• 6.6. The inhibition by Fru-2,6-P₂ is released by Mg²⁺ and more powerfully by Mn²⁺ increasing the I_0.5.

     

Potassium channels in growth cones of leech retzius cells

• 1.1. Potassium-selective channels were analysed in growth cones of cultured leech Retzius cells.
• 2.2. In the cell-attached mode and at physiological bath and pipette solution little channel activity was observed at resting membrane potential. The channel open probability (p_o) increased with cell depolarization, and the slope conductance of the single K⁺ channel current was about 60 pS.
• 3.3. With symmetrical high KCl solution on both sides of the excised membrane patch three K⁺ -selective channels could be discriminated. Two channels exhibited a linear current-voltage relation of about 18 pS and 106 pS, respectively.
• 4.4. The most frequently observed K⁺ channel showed a non-linear current-voltage relation and p_o increased with increasing free cytoplasmic Ca²⁺ and during cell hyperpolarization.

     

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique

• 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
• 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
• 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
• 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (K_a) of 2.36 × 10⁷ and 3.36 × 10⁶M⁻¹.
• 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

An in vitro study of short-chain fatty acid concentrations,production and absorption in pig (Sus scrofa) colon

• 1.1. Short-chain fatty acid concentration was 180mmol/l in the proximal colon and decreased to 108 mmol/l in the rectum.
• 2.2. Fermentation in chymus from different regions of the colon, showed the pattern of end products to reflect the substrate and not the site of the colon.
• 3.3. Isolated mucosa from proximal and distal colon had electroneutral sodium absorption of 4.8 ± 0.2 and 2.9 ± 0.8 μeq/cm² hr in bicarbonate free media, which was abolished in the absence of chloride.
• 4.4. Electroneutral sodium absorption was enhanced by short-chain fatty acids in the proximal colon and could be described by Michaelis-Menten kinetics with K_m 2.0–11 mmol/l and J_m 1.6–3.6μeq/cm² hr. In the distal colon the stimulation was smaller and propionate even inhibited sodium absorption.
• 5.5. Butyrate was absorbed in the proximal colon, whereas acetate and propionate, and butyrate in the distal colon had a flux ratio of one.
• 6.6. Amiloride (5 mmol/l) inhibited sodium absorption and net butyrate absorption.

     

Mechanisms of membrane potential oscillation in bursting neurons on the snail,Helix pomatia

• 1.1. The mechanism of generation of membrane potential (MP) oscillations was studied in identified bursting neurons from the snail Helix pomatia.
• 2.2. Long-lasting stimulation of an identified peptidergic interneuron produced a persistent bursting activity in a non-active burster.
• 3.3. External application of calcium channel blockers (1 mM Cd²⁺ or 5 mM La²⁺) resulted in a transient increase in the slow-wave amplitude and subsequent prevention of pacemaker activity generation in bursting neurons. Application of these blockers together with endogenous neuropeptide initiating bursting activity generation, increased MP wave amplitude without prevention of bursting activity generation.
• 4.4. Replacement of all NaCl in normal Ringer's solution with isoosmotic CaCl₂, glucose or Tris-HCl produced a reversible block of bursting activity generation. Stationary current-voltage relation (CVR) of bursting neuron membrane has a region of negative resistance (NRR) and does not intersect the potential axis in threshold region for action potential (AP) generation in normal Ringer's solution. In Na-free solution stationary CVR is linear and intersects the potential axis near — 52 mV.
• 5.5. Novel potential- and time-dependent outward (E_rev = − 58 mV) current, I_B, activated by hyperpolarization was found in the bursting neuron membrane. Having achieved a maximal value, this current decayed with a time constant of about 1 sec. Hyperpolarization inactivated maximal conductance, g_B, responsible for I_B, and depolarization abolished inactivation of g_B.
• 6.6. Short-lasting (0.01 sec) hyperpolarization of the bursting neuron membrane by inward current pulse induced the development of prolonged hyperpolarization wave lasting up to 10 sec.
• 7.7. These results suggest that: (a) persistent bursting activity of RPal neuron in the snail Helix pomatia is not endogenous but is due to a constant activation of peptidergic synaptic inputs of these neurons; (b) Ca²⁺ ions do not play a pivotal role in the ionic mechanism of MP oscillations but play a determining role in the process of secretion of a peptide initiating bursting activity by the interneuron presynaptic terminal; (c) depolarizing phase of the MP wave is due to specific properties of stationary CVR and hyperpolarization phase is due to regenerative properties of hyperpolarization-activated outward current I_B. The minimal mathematical version of MP oscillations based on the experimental data is presented.

     

Identification of essential amino acids in the active center of thyroidal NAD+ glycohydrolase

• 1.1. Purified thyroidal NAD⁺ glycohydrolase has been subjected to the action of a number of group specific reagents in order to gain information concerning its mode of action.
• 2.2. Modification of histidyl residues with diethylpyrocarbonate strongly suppresses the NAD⁺ glycohydrolase activity. Inactivation with this reagent can be reversed to some extent by subsequent treatment with hydroxylamine.
• 3.3. NAD⁺ and ADP-ribose partially protect against inactivation with similar efficiencies.
• 4.4. The incomplete reactivation with hydroxylamine after diethylpyrocarbonate treatment and the selective inactivation by 2,4-pentanedione indicates that apart from one or more essential histidyl residue(s) also lysyl residues are important for activity. NAD⁺ and to a smaller extent ADP-ribose again protect against inactivation by 2,4-pentanedione.
• 5.5. The sensitivity of the enzyme towards N-ethyl-5-phenyl-isooxazolium-3'-sulfonate further points to the importance of carboxylate containing side chains.
• 6.6. The mechanistic implications of these results are discussed.

     

Thermal properties for digestive enzymes of a sub-antarctic beetle,hydromedion sparsutum (coleoptera,perimylopidae) compared to those in two thermophilic insects

• 1.1. Proteolytic, lipolytic, amylolytic and cellulolytic activities were studied in adults of the phytophagous beetle, Hydromedion sparsutum, indigenous to the sub-Antarctic island of South Georgia.
• 2.2. Gastric enzyme activities were measured at experimental temperatures of 5–40°C and results were compared with those obtained from two thermophilic insects, Gryllus bimaculatus and Tenebrio molitor.
• 3.3. Protease and lipase activities in Hydromedion were 10–15 times lower than in Gryllus and Tenebrio.
• 4.4. In the temperature range of 5–15°C, α-amylase activity from Hydromedion was only slightly lower than that from Gryllus.
• 5.5. Hydromedion gut homogenates exhibited a distinct cellulolytic activity, even at a low temperature of 5°C.
• 6.6. Cellulolytic activity in the digestive tract of Hydromedion was confirmed by the evolution of ¹⁴CO₂ after consumption of labelled cellulose.
• 7.7. The thermal properties of digestive enzymes agree well with the role of Hydromedion as primary decomposer in its ecosystem.

     

Demonstration of highly specific and sensitive antibodies to a naturally occurring tetrahydroisoquinoline alkaloid,salsolidine

• 1.1. We report for the first time on the production and characterization of antibodies against a naturally occurring tetrahydroisoquinoline, namely salsolidine (6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline).
• 2.2. Immunogen synthesis was carried out by coupling the hapten salsolidine to bovine serum albumin (BSA) as carrier protein on the basis of reductive amination.
• 3.3. By immunization of rabbits with salsolidine-BSA conjugate antisalsolidine antibodies were produced.
• 4.4. At a final dilution of 1:1700 the highest-litre antiserum bound 35% of 0.21 pmol [³H] salsolidine. This antiserum was used to develop a radioimmunoassay for salsolidine.
• 5.5. Cross-reactivity studies revealed a high specificity of the antiserum to the hapten.
• 6.6. The antibodies had a high affinity to salsolidine (K_a = 1.5 × 10⁹ M⁻¹).
• 7.7. Standard curves covered a measuring range of 0.5–70 pmol/tube and the detection limit was found to be 0.27 pmol/tube.

     

Acute toxicity and bioaccumulation of endosulfan in rotifer (Brachionus calyciflorus)

• 1.1. The acute toxicity of endosulfan was determined for the freshwater rotifer Brachionus calyciflorus.
• 2.2. The mean 24 hr lc₅₀ value for endosulfan was 5.15 ppm with a coefficient of variation of 14.7%.
• 3.3. Rotifers were exposed at two sublethal concentrations (1.5–2.0 ppm) of endosulfan for bioaccumulation experiments, for an exposure time of 24, 48, 72 and 96 hr. The rotifers were fed with Nannochloris oculata (5 × 10⁵cell/ml).
• 4.4. The highest accumulation of endosulfan was found 24 hr after the start of the exposure to 1.5 ppm of the toxicant. A steady-state concentration in rotifer was reached between 24–48 hr, followed by a gradual decrease until 96 hr.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Purification and some properties of a Mg2+-activated acid phosphatase from rat testis

• 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
• 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
• 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
• 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
• 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.

     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

Prostaglandin E1 and analogs of prostacyclin influencing superoxide production by the human neutrophil nadph oxidase system

• 1.1. The effects of prostaglandin (PG) E₁, and I₂ analogs (OP-41483 and OP-2507) on the Superoxide generation of human neutrophil NADPH oxidase (EC 1.6.99.6) in both whole-cell and cell-free systems were investigated.
• 2.2. In a whole-cell system, OP-2507 inhibited the Superoxide generation by neutrophils exposed to phorbol myristate acetate concentration-dependently through its superoxide-scavenging action.
• 3.3. The concentration of the drug required for 50% inhibition of the oxidase (IC₅₀) was 21 μM.
• 4.4. In a cell-free system, however, the drug in concentrations of < 100 μM did not inhibit the activation of NADPH oxidase by sodium dodecyl sulfate because of its inactivation by the detergent.
• 5.5. Although PGE₁ and OP-41483 did not inhibit the Superoxide production by stimulated neutrophils in a whole-cell system, they both inhibited the activation of NADPH oxidase in a cell-free system concentration-dependently, with IC₅₀ values of 44 and 170 μM, respectively.
• 6.6. In addition, in the cell-free system, the K_m value for NADPH of the oxidase was unchanged by PGE₁.
• 7.7. The results suggest that the PGI₂ analog, OP-2507, is a possible superoxide-scavenger and that PGE₁ inhibits the NADPH oxidase activation by sodium dodecyl sulfate in a cell-free system concentration-dependently.

     

Effect of experimental ventilation of the skin on cutaneous oxygen uptake in the carp,Cyprinus carpio

• 1.1. Cutaneous O₂ uptake in the carp, Cyprinus carpio, was determined at various water flow rates across the skin (.V) ranging from 2.5 to 40 ml/min, using flow-through respirometers.
• 2.2. When thickness of water flow was 2mm, cutaneous O₂ uptake remained stable (about 3.8 nmol/cm²/min) at a .V of 20–40 ml/min and decreased with .V below 20 ml/min.
• 3.3. When thickness of water flow was 4 mm, cutaneous O₂ uptake decreased with .V below 40 ml/min.
• 4.4. Apparent water velocity (U') was calculated dividing .V by an area of a cross section of the water flow (0.5 and 1.0 cm² respectively). In both experiments, cutaneous O₂ uptake decreased with U' below 0.7 cm/sec.
• 5.5. This suggests that cutaneous O₂ uptake in the carp is limited at a low water velocity by a resistance of the hypoxic boundary layer.