Enhancement of erythrophagocytosis by cyclophosphamide

The effect of cyclophosphamide (Cy) on erythrophagocytosis carried out by peritoneal exudate cells (PEC) was evaluated. The results indicate that the treatment of BALB/c mice with a single high dose of Cy (200 mg/kg) increases the capability of PEC to ingest sensitized sheep red blood cells. Cy not only increases the percentage of PEC with engulfed erythrocytes, but also augments the activity of individual phagocytes. This effect is exerted upon both mononuclear and polymorphonuclear cells and is probably due to the activation of receptors for the Fc fragment of IgG on PEC membranes, as unsensitized sheep erythrocytes are not phagocytized. Considering that Cy is one of the most used alkylating agents in treatment of immunological disorders and cancer chemotherapy, this enhancing effect on phagocytosis may be important in the regulation of host defense mechanisms.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.     

Generation of superoxide anion and hydrogen peroxide induced by nifurtimox in Trypanosoma cruzi.

Addition of nifurtimox (a nitrofuran derivative) to Trypanosoma cruzi culture (epimastigote) forms induced an increase in the respiratory rate and the release of H₂O₂ from the whole cells to the suspending medium. Growth-inhibiting concentrations of nifurtimox were able to stimulate O_2? production by the T. cruzi mitochondrial fraction supplemented with NADH (or succinate), and also to enhance the generation of O_2? by the microsomal fraction with NADPH as reductant.     

Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient

A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 μm pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between δ1.050 and δ1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia.A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera.The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.     

Effects of prostaglandins E2, I2 and F2α, arachidonic acid and indomethacin on pressor responses to norepinephrine in conscious rats

The effects of prostaglandins E₂ (PGE₂), I₂ (PGI₂) and F₂α (PGF₂α), arachidonic acid and indomethacin on pressor responses to norepinephrine were examined in conscious rats. Intravenously infused PGE₂ (0.3, 1.25 μg/kg/min), PGI₂ (50, 100 ng/kg/min), PGF₂α (1.8, 5.4 μg/kg/min) and arachidonic acid (0.7, 1.4 mg/kg/min) did not change the basal blood pressure. Both PGE₂ and PGI₂ significantly attenuated pressor responses to norepinephrine, whereas PGF₂α significantly potentiated them. Arachidonic acid, a precursor of the prostaglandins (PGs), significantly attenuated pressor responses to norepinephrine. Since the attenuating effect of arachidonic acid was completely abolished by the pretreatment with indomethacin (5 mg/kg), arachidonic acid is thought to exert an effect through its conversion to PGs. On the contrary, intravenously injected indomethacin (0.2–5.0 mg/kg) facilitated pressor responses to norepinephrine in a dose-related manner without any direct effect on the basal blood pressure. These results suggest that endogenous PGs may participate in the regulation of blood pressure by modulating pressor responses to norepinephrine in conscious rats.     

The effect of 3-mercaptopicolinic acid on phosphoenolpyruvate carboxykinase (GTP) in the rat and guinea pig.

In vitro, 3-mercaptopicolinic acid inhibited phosphoenolpyruvate carboxykinase activity in supernatant fractions of liver, kidney cortex, and adipose tissue obtained from fasted rats. 3-Mercaptopicolinic acid also inhibited enzymatic activity in the mitochondrial and supernatant fractions of liver obtained from fasted guinea pigs. In the fasted rat, the oral administration of 3-mercaptopicolinic acid increased liver carboxykinase activity even though the blood glucose concentrations decreased. Kidney cortex carboxykinase decreased while adipose tissue enzyme was unchanged. In the fasted guinea pig, the oral administration of 3-mercaptopicolinic acid lowered blood glucose concentrations but had no effect on liver mitochondrial or supernatant carboxykinase activity. The elevation in rat liver enzymatic activity appears to be due to protein synthesis, since the concurrent administration of cycloheximide prevents the increase in enzyme activity. 3-Mercaptopicolinic acid appears to be noncompetitive with respect to Mn²⁺.     

Evidence of essential arginyl residues in rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-first-order kinetics with a calculated second-order rate constant of 4.6 m^?1 min^?1. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified; this result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule per subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K⁺ and Mg²⁺, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate.     

Studies on the mode of action of calciferol. XVIII. Evidence for a specific high affinity binding protein for 1,25 dihydroxyvitamin D3 in chick kidney and pancreas.

Cytosol fractions prepared from rachitic chick kidney and pancreas were analyzed for binding of vitamin D₃ metabolites by sucrose density gradient centrifugation. Both cytosol fractions were found to contain a 3.6S macromolecule which specifically binds 1,25-dihydroxy[³H] vitamin D₃ and in addition a 5 to 6S macromolecule which binds 25-hydroxy[³H]vitamin D₃. Sucrose gradient analysis of a KCl extract prepared from kidney or pancreas chromatin resulted in a peak (3.6S) of bound 1,25-dihydroxyvitamin D₃ which could not be distinguished from the cytoplasmic binding component. The interaction of 1,25-dihydroxy[³H]vitamin D₃ with the cytoplasmic binding component of both tissues occurred at low concentrations of hormone with high affinity.     

Correlations among the responses of suspensions of Dictyostelium Discoideum to pulses of 3′,5′-cyclic AMP: Transient turbidity decrease,the cyclic AMP signal,and variation in adenine mononucleotide levels

Aggregation-competent myxamoebae of the cellular slime mold Dictyostellium discoideum are known to exhibit two responses to extracellular pulses of 3′5′-cyclic AMP: an immediate chemotactic movement; and a delayed generation of intracellular cyclic AMP which is subsequently released into the medium. The mechanism of the latter, the so-called signalling response, may depend on alterations in intracellular metabolite levels and is the subject of this communication.Myxamoebae of the wild-type strain NC-4 of D. discoideum were suspended in an aerated, stirred 17 mM potassium phosphate buffer. pH 6.0, at a concentration of approx. 6 · 10^?7 cells/ml (8%, v/v) at 25°C and were pulsed with 1. 10^?8—1 · 10^?7 M cyclic AMP at 10–20-min intervals for periods of 3–5 h over incubation of 4–9 h. Suspensions were monitored continuously for transient turbidity decreases following the cyclic AMP pulses as an indication of the magnitude and duration of the cellular response to cyclic AMP. When the pattern of turbidity decrease indicated that a signalling response had developed, samples were withdrawn at 10–15-s intervals from the suspension, inactivated with perchloric acid, and analyzed for cyclic AMP, ATP, ADP, AMP, pyruvate, and glucose 6-phosphate. In separate experiments, steady-state oxygen tension was monitored along with turbidity to detect possible changes in respiratory rate.The following consistent patterns were observed after the added cyclic AMP pulse: a transient increase in the ADP level which reaches maximum between 0.7 and 1.7 min; transient decreases in ATP and pyruvate which concide with and approximately equal the magnitude of the increase in ADP; a later increase in glucose 6-phosphate which reaches maximum approx. 2 min after the ADP     

Measurement of plasma α-ketoisocaproate concentrations and specific radioactivity by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic technique for the measurement of the specific radioactivity and concentration of α-ketoisocaproate in plasma is described. Plasma proteins are precipitated by additions of acetone, the supernatant is applied to a cation-exchange column, and the resulting eluate is injected into a C₁₈ reverse-phase column. Analysis time is approximately 10 min. Quantitative recovery, specificity, and sensitivity of this method are described and make this system attractive for in vivo α-ketoisocaproate kinetic studies. Using this procedure, the apparent flux of α-ketoisocaproate in postabsorptive dogs was determined during an infusion of α-[U-¹⁴C]ketoisocaproate and averaged 2.8 + 0.41 μmol/kg-min.     

Pharmacological evidence that LH-RH action on lordosis behavior is mediated through a rise in cAMP

The effect of two phosphodiesterase inhibitors, theophylline (THP) and 1-methyl-3-isobutyl-xanthine (MIX), on the lordosis response induced by three dose levels (0.5, 1, and 5 μg) of LH-RH was studied in ovariectomized estrogen-primed rats (estradiol benzoate, 4 μg). Neither THP (10 mg) nor MIX (2mg) facilitated lordosis behavior in estrogen-primed rats. Combined administration of 10 mg of THP with LH-RH exerted only a slight facilitatory effect on lordosis behavior. Administration of 2 mg of MIX significantly synergized with LH-RH for the facilitation of lordosis. The results suggest that LH-RH elicits sexual behavior by increasing cAMP levels in neurons related to the expression of lordosis behavior.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

Effect of castration and testosterone on norepinephrine storage and on the release of [3H]norepinephrine from rat vas deferens

Norepinephrine and dopamine-β-hydroxylase, used as noradrenergic vesicle markers, were found to be decreased in the rat vas deferens 10 days after castration. Five days of testosterone administration to castrated animals increased the enzyme activity over that of controls but did not modify norepinephrine content. In tissue fractions obtained by differential centrifugation, the highest activities of the noradrenergic markers appeared in the vesicular fraction of controls and in the soluble fraction of castrated animals. Testosterone reversed the effect of castration: it increased dopamine-β-hydroxylase activity in the vesicular and soluble fractions, while norepinephrine increased only in the vesicular fraction. Results obtained after continuous sucrose gradient centrifugation of vesicular fractions suggested that these changes principally affected the number of light noradrenergic vesicles while testosterone increased the number of vesicles reduced by castration. Hormonal manipulations also modified some functional properties of nerve endings: norepinephrine depletion after transmural stimulation in the presence of tetraethylammonium, as well as the release of the neurotransmitter, were decreased after castration. These effects were reversed by testosterone. The results suggest a modulatory effect of testosterone on the norepinephrine storage system and on the functional properties of the adrenergic innervation of vas deferens.     

A reaction between the superoxide free radical and lipid hydroperoxide in sodium linoleate micelles

The oxidation of unsaturated fatty acid micelles by the superoxide free radical ($\text{O}{}^{\mathrm{?}}{}_2$), during γ irradiation in the presence of formate, is kinetically distinct from oxidation by hydroxyl free radicals (HO.). The evidence suggests that a direct reaction between ($\text{O}{}^{\mathrm{?}}{}_2$) and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with ($\text{O}{}^{\mathrm{?}}{}_2$), protects the micelles from oxidation, active superoxide dismutase is no more effective than its apoprotein, due to lack of penetration of the micellar environment. We discuss these findings in the light of recent literature, and with reference to their possible significance for biological systems.     

Chemical intermediates in dopamine oxidation by tyrosinase,and kinetic studies of the process

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H⁺ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine → o-dopaminequinone-H⁺ → o-dopaminequinone → leuko-dopaminochrome → dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H⁺ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H⁺ → dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E_ZCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H⁺ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.     

Interaction of thyroid hormone and sex steroids at the rabbit reticulocyte membrane in vitro: control by 17 beta-estradiol and testosterone of thyroid hormone-responsive Ca2+-ATPase activity

Physiological concentrations (10(-10) M) of L-thyroxine and triiodo-L-thyronine were found in vitro to enhance Ca2+-ATPase activity in reticulocyte-enriched red cell membranes from female rabbits and to inhibit this enzyme in the male reticulocyte. Cross-incubation experiments with reticulocyte-enriched red cells and plasma from the opposite sex demonstrated that this sex-specific membrane response to thyroid hormone was transferable by plasma. Similar experiments with intact reticulocytes exposed to physiological concentrations (10(-11) M) of testosterone and 17 beta-estradiol indicated that the plasma factors were the sex steroids. That is, incubation in vitro with testosterone converted female-source reticulocytes to male-type responsiveness to thyroid hormone (inhibition of Ca2+-ATPase activity); incubation with estradiol converted male-source reticulocyte-enriched red cells to female-type responsiveness (stimulation by iodothyronines of membrane Ca2+-ATPase activity). Similar results were obtained when reticulocyte ghosts were incubated with testosterone and 17 beta-estradiol prior to determination of membrane enzyme activity. Etiocholanolone (5 beta-androstan-3 alpha-ol-17-one) and testosterone were equipotent, but 5 alpha-dihydrotestosterone had little activity in this system. Estrone and estradiol were equipotent, but estriol had no permissive effect on the stimulation by iodothyronine of reticulocyte membrane Ca2+-ATPase activity. Expression of thyroid hormone action in vitro on Ca2+-ATPase activity in the rabbit reticulocyte is determined at the membrane level by testosterone and estrogen. The structure-activity relationships of the sex steroids for this membrane action are different than those reported for nuclear actions of the steroids.     

Regulation of cyclic GMP, cyclic AMP and lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line.

In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a β-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of α-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.