Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

The effect of prostaglandins A2,E1,E2,15 methyl E2, 16, 16 dimethyl E2 and F2α on erythropoiesis

Prostaglandins A₂, E₁, E₂, methylated E₂s and F₂α affected erythropoiesis and/or erythropoietin (Ep) production. This action is indicated in the exhypoxic, polycythemic mouse where radioiron incorporations into RBC increased after administration of these compounds. The kidney and liver have been indicated through previous studies, to actively participate in Ep production. By the removal of one of these active sites in a murine system treated with prostaglandins it is shown that a response is reflected in Ep levels. Interference of the action of prostaglandins (PG) is altered by the removal of one of these target sites of Ep production. The erythropoietic responses elicited by PGA₂, E₁, and perhaps the methylated PGE₂s act through the liver whereas PGE₂ may operate through a renal pathway for its response. PGF_2α reveals no effect on erythropoietic activity and is no different than that observed for vehicle-treated controls. The prostaglandins tested appear to act primarily through the kidney or liver but the possibility exists that some yet undetermined organ site may also be involved.     

The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides.

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.     

The reciprocal exclusion by L-dopa (L-3,4-dihydroxyphenylalanine) and L-tyrosine of their incorporation as single units into a soluble rat brain protein.

Several compounds, structurally and metabolically related to phenylalanine and tyrosine, were tested for their effects on the incorporations of phenylalanine and tyrosine as single units into a protein of the soluble subcellular fraction of rat brain. Of the compounds tested, only L-dopa (L-3,4-dihydroxyphenylalanine) inhibited these incorporations. Further, L-dopa was incorporated into a protein of the same fraction in such a way that it excluded the incorporation of tyrosine as a single unit. Conversely, tyrosine inhibited and excluded the incorporation of L-dopa. The incorporation of L-dopa required ATP (apparent Km = 0.23mM), KCl (apparent Km = 20mM) and MgCl2 (optimal concentration range, 5-16mM). These requirements were similar to those previously determined for the incorporation of tyrosine and phenylalanine. The inactivation rate of the enzymic systems for L-tyrosine and L-dopa incorporations, when kept at 37 degrees C, was the same for both amino acids (half-life = 80 min). It is suggested that the acceptor for the incorporation of dopa is the same as that for the incorporation of tyrosine.     

Effects of cytoprotective antioxidants on lymphocytes from representative mitochondrial neurodegenerative diseases

Two new aza analogues of the neuroprotective agent idebenone have been synthesized and characterized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scavengers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol derivatives were also shown to augment ATP levels in coenzyme Q₁₀-deficient human lymphocytes. The more lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibition of the electron transport chain and improved antioxidant activity.     

Communication interference in sympatrically occurring moth species

In moth species, females emit a species‐specific sex pheromone that is perceived over long distance by conspecific males. The species‐specificity in the chemical communication channel is achieved by a combination of unique components in specific ratios and sometimes also by interspecific behavioural antagonists to deter sympatrically occurring heterospecific males. In this study, we determined possible antagonistic effects in Helicoverpa gelotopoeon Dyar (Lepidoptera: Noctuidae) males to the major sex pheromone component of sympatrically occurring heliothine moths, Z11‐16:Ald, as well as to the sex pheromone of the sympatrically occurring Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) (Z11‐16:Ald and Z9‐14:Ald). We also explored whether other co‐occurring species are attracted to these pheromone blends. Our field experiments showed that the addition of Z11‐16:Ald alone or in combination with Z9‐14:Ald inhibited trap catches of H. gelotopoeon males and that this inhibition depended on the concentration of these compounds. In addition, other moth species were attracted to the blends. Together, our results confirm the antagonistic effect of heterospecific sex pheromone compounds of H. virescens to H. gelotopoeon.     

Tubulin–Na+ ,K +-ATPase interaction: Involvement in enzymatic regulation and cellular function

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na⁺,K ⁺-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin–NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na ⁺. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na ⁺ and K ⁺, and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na ⁺/K ⁺ homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.     

Effect of cation and anion of an electrolyte on apparent molar volume, isentropic compressibility and refractive index of glycine in aqueous solutions

Experiments at 298.15 K have been performed to measure the density, velocity of sound and refractive index in three water+glycine+electrolyte systems. The electrolytes studied were KCl, KNO3 and NaNO3. The values of apparent molar volume and isentropic compressibility of glycine in aqueous electrolyte solutions were calculated from the measured data. The results obtained in this study and those reported previously for water+glycine+NaCl system have been comparatively studied. The results show that the nature of both the cation and the anion of an electrolyte influence the behaviour of glycine in aqueous solutions. For all four electrolytes studied, the comparison shows a positive volume transfer for glycine from an electrolyte solution to a more concentrated solution of the same electrolyte. The results also show a negative apparent isentropic compressibility for glycine in the presence of the electrolytes studied. These effects indicate that the volume of a glycine molecule is larger in solutions with higher electrolyte concentration and the water molecules around the glycine molecules are less compressible than the water molecules in the bulk solution. These effects were attributed to the doubly charged behaviour of glycine and to the formation of physically bonded ion-pairs between the charged groups of glycine and the cation and the anion of the electrolyte.     

Autoradiographic localization of angiotensin II receptors in developing rat cerebellum and brainstem

The role of Angiotensin II (Ang II) as a growth promoting or modulating factor has recently become a field of intensive research. A central issue in developmental neurobiology is the understanding of mechanisms governing the formation of spatially ordered connections. In this study, we show the localization of Ang II receptor subtypes by autoradiography in 2-week-old rat hindbrains confronting these data with membrane binding assays. Competition studies done on membrane preparations evidence no major changes on the relative affinities for both receptor subtypes between 2-week-old and adult rat tissues. By autoradiography, we found that all the areas (1-10) of the 2-week-old cerebellum showed both receptor subtypes present in complementary adjacent layers. Areas expressing a high level of AT2 receptors follow: inferior colicullus (IC), dorso tegmental nucleus, central (DTgC), subcoeruleus, alpha, sensory root of the trigeminal nerve, principal sensory root trigeminal nucleus (Pr5, Pr5VL) supragenual nucleus, genu facial nerve, facial nucleus, cerebellar peduncles, vestibular and lateral nuclei. Spinal trigeminal, (oral) and Raphe nuclei express AT1 receptor subtype. The high level of Ang II AT2 receptors present in the cerebellar peduncles might have a meaning on the establishment of the olivo-cerebellar connection. The high expression of Ang II AT2 receptors on 2-week-old rat hindbrains, a critical age on development, as well as its disappearance in the adult, strongly suggests a probable role of these receptors in cell migration and neuronal synaptogenesis.     

Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within approximately 48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1 alpha), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-gamma), CXCL10 (IFN-gamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.