High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis

Summary A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F₁ progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment.Contribution No. 89-524-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan     

1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes.

The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

The biological half life and distribution of 125Iodide and radioiodinated protein in the imported fire ant, Solenopsis invicta

The radioisotope ¹²⁵Iodide, a gamma emittor, was used in two different forms, as ¹²⁵I mixed with egg yolk and as ¹²⁵I covalently attached to egg albumin and mixed with egg yolk, to study food flow in the imported fire ant, Solenopsis invicta Buren. The biological half life of ¹²⁵I-albumin in egg yolk powder was determined to be 96 hr in isolated workers, 108 hr in individuals held with small groups of unlabelled workers, and 1,008 hr in workers held in colonies exposed to labelled food for 48 hr. In contrast, the biological half life of free ¹²⁵I mixed with egg yolk powder was 22 hr, 20 hr, and 40 hr, respectively.The internal distribution of radioactivity was checked after 24,48, and 380 hr. There was a significant difference in distribution of ¹²⁵I in ants fed either free ¹²⁵I or ¹²⁵I-albumin. Most of the free ¹²⁵I was rapidly excreted. A high percentage of ¹²⁵I-albumin was assimilated, apparently through protein digestion pathways with eventual storage in or below the cuticle. There was no evidence of gland involvement in food flow to either larvae or queens with the radio-iodinated protein.

---

Résumé L'utilisation de l'iode radio-actif (¹²⁵I) a permis d'étudier le cheminement de la nourriture chez Solenopsis invicta Buren (Myrmicinée). Deux formes différentes de l'isotope ont été étudiées. L'iode 125 a été fixé d'une manière covalente à la tyrosine dans l'albumine des oeufs en utilisant la méthode chloramine T pour ioder les protéines. L'albumine marquée a été mangée ensuite à du jaune d'oeuf en poudre.La seconde forme contenait de l'iode 125 mélangé au jaune d'oeuf en poudre en absence de tout catalyseur, ce qui empêche la fixation chimique. La demi-vie biologique (T_biol) des deux formes a été déterminée chez des ouvrières isolées, chez des individus gardés avec de petits groupes d'ouvrières non-marquées, et chez des ouvrières gardées dans des colonies exposées à la nourriture radio-active pendant 48 h. La demi-vie biologique de l'albumine marquée était de 96 h, 108 h, et 1.008 h. En contraste, la demi-vie de l'iode 125 était de 22 h, 20 h, et 40 h. L'effet de groupe créé par des échanges répétés de nourriture entre les individus était négligeable avec la nourriture protéique. L'échange répété de nourriture entre les larves et les ouvrières a beaucoup augmenté la demi-vie de l'albumine marquée à I 125. Cet effet n'était pas aussi clair avec l'iode 125 par suite de son élimination rapide.La distribution de la radio-activité a été examinée chez des ouvrières au bout de 24, 48 et 380 h après les avoir nourries avec de l'albumine marquée à I 125 et de l'iode 125. Il y avait une différence considérable de distribution, avec un haut pourcentage d'albumine assimilé, sans doute par les voies de digestion de protéines. Le radio-isotope a été ensuite conservé sous forme d'iode 125 ou d'iodotyrosine, dans (ou sous) la cuticule de l'ensemble du corps. Les fourmis ont rapidement excrété l'iode 125 libre avec 5% de la radioactivité résiduelle après 380 h, peut-être fixée aux protéines cellulaires, et ensuite transportée vers la cuticule. Les différences considérables entre les demi-vies biologiques et la distribution interne de la radioactivité chez les fourmis nourries avec de l'iode 125 ou à l'albumine marquée à I 125, soulignent le danger de croire que le cheminement de la nourriture peut être définitivement étudié en utilisant des radio-isotopes qui ne sont pas fixés chimiquement à la substance étudiée.

     

The labial gland system of larvae of the imported fire ant,Solenopsis invicta Buren

Summary The authors describe the ultrastructure of the labial gland system in imported fire ant larvae, Solenopsis invicta Buren and present an enzyme analysis of enzymes in labial-gland secretions and midgut contents. The tubes of the labial gland produce and secrete a proteinaceous substance rich in digestive enzymes. The narrow cells of the reservoir of the gland have little or no secretory function but the lumen stores the proteinaceous secretion. The labial-gland enzymes include proteases and amylases, which function in extraintestinal digestion of solid food placed on the anteroventral body region of 4th-instar larvae by adult workers. The midgut contains proteases, amylases, and upases. Lipids appear to be predigested by the workers before being fed to the larvae.Approved as TA 15262 by the Director of the Texas Agricultural Experiment Station in cooperation with ARS/USDA. Supported by the Texas Department of Agriculure interagency agreement IAC-0487 (78-79)We would like to thank Rosemary Kamas, Dr. John Mirenda, and Mike Strand for their help in dissections of larvae and Dr. Howard Williams for his advice and suggestions     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the brain and infundibulum of the sheep

Summary To determine how neural influences control the function of the pineal gland, morphological and biochemical relationships after pharmacological treatment have been studied in rat pineal cells in monolayer cultures. Norepinephrine (NE) and dibutyryl cyclic 3,5-adenosine monophosphate (dBcAMP) treatment of cells that had been in culture for 5 and 21 days produced a stimulation in the enzyme activity of serotonin N-acetyl transferase, an enzyme important in indole synthesis. NE and dBcAMP also produced morphological changes which were dependent on the time of cells in culture. When 5 day-cultures were treated with NE and dBcAMP, light and dark cells were noted and endoplasmic reticulum increased and became more organized. Only dBcAMP treatment at 5 days produced an increase in dense granules and an elongation of cytoplasmic processes. Treatment of 21 day-cultures with dBcAMP also produced an increase in cytoplasmic processes while treatment with NE produced an increase in the synaptic ribbons and clear vesicles within the processes.     

Isolation of an aggregation inhibitory factor from non-adhesive mouse teratoma cells

An aggregation inhibitory factor (AIF) has been extracted from mouse ascites teratoma cells (that do not aggregate in culture) that retards adhesion of cultured teratoma cells of the same cell line (that do aggregate). Preliminary characterization of AIF on polyacrylamide gels suggests that AIF is a protein composed of four subunits. Extraction of AIF from ascites teratoma cells was accomplished without significant loss of viability by a technique involving the application of an electric field to large numbers of whole cells suspended in a hypertonic electrode buffer. In tests of adhesion, AIF consistently and immediately inhibited aggregation of cultured teratoma cells after 5, 10, 15, and 30 min of incubation. Furthermore, a reduced concentration of AIF resulted in a corresponding decrease in inhibition, suggesting a concentration-dependent action. AIF may help explain how cultured teratoma cells adhere, whereas ascites teratoma cells of the same subline do not adhere.     

Inhibition of hydroxyproline synthesis by palladium ions.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.