On the ultrastructure and the supposed function of the mineralizing matrix coat of sea urchins (Echinodermata,Echinoida)

Summary Echinoderm ossicles are part of the mesenchyme. Their formation and growth, with respect to the underlying tissues, is studied using echinoid spines and teeth and applying different methods of fixation. The calcification process in echinoderms is strictly intracellular and needs (1) syncytial sclerocytes which completely enclose (2) a vacuolar cavity which in turn contains (3) an organic matrix coat. Strictly speaking, each ossicle is nothing but the calcified vacuolar space of a single syncytium of sclerocytes. In fully grown parts, however, the continuous sheath may split open and the matrix-coated mineral may come into contact with the extracellular space. According to biochemical analyses the matrix consists of insoluble components, but most (95%) of its constituents are soluble in EDTA or weak acids. If routine transmission electron microscope methods are used the soluble components are lost and the matrix at best looks electron light. If tannic acid is added to the fixative the soluble matrix components are preserved and reveal further ultrastructural details of the biomineralization process in echinoderms. The matrix coat looks extremely electron dense. Further soluble material is to be found within the vacuolar space or attached to the vacuolar surface of the cytoplasmic sheath. The results lead to the opinion that the matrix coat consists of a hydrophobic framework of insoluble components that contains soluble components which guide the Ca through pores in the hydrophobic layers into the interior of the matrix-coated space. It is only within this space that the mineral is deposited.     

Effects of temperature on cytomorphogenesis and ultrastructure ofMicrasterias denticulata Bréb

Summary Exposure of growingMicrasterias cells to high (32°–36°C) and low (3°–10°C) temperatures produces changes in morphology that are accompanied by several ultrastructural alterations. Whereas low temperatures essentially cause simplification of cell ornamentation, a variety of cell malformations result from high temperature treatment. These are the loss of cell symmetry leading to markedly aberrant cell shapes and an increase of main lobes with reduced degree of differentiation. Preliminary studies indicate that a shift in the distribution of membrane-associated Ca²⁺ by elevated temperatures probably underlies these abnormal cytomorphogenetic events. Both, low and high temperature cause a reduction in size of the young half cell and affect cytoplasmic streaming. Moreover, nuclear migration is retarded and chloroplast arrangement is influenced by temperature treatment at both ranges. Growth velocity of primary wall responsible for cell shaping is increased at high and slowed down at low temperatures compared to cells grown at 20°C.The main ultrastructural alterations induced by high temperatures are an increase in amount and length of ER cisternae, the appearance of heat shock granule aggregations localized in the cytoplasm, a reduced number of ribosomes and polysomes, the presence of oil bodies in growing cells and a varying thickness of the primary wall. Influences of low temperatures on ultrastructure are less pronounced. They are manifested in the formation of large aggregations of ER cisternae slightly differing from those found in untreated cells, a disturbed arrangement of the microtubule system surrounding the nucleus and a decrease of the number of cell wall forming cytoplasmic vesicles.It is thought that most of the temperature effects are due to an influence on membranes probably an alteration of ionic currents and, in addition, a modulation of normal protein synthesis.Dedicated to my teacher Professor Oswald Kiermayer in deep gratitude     

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.     

The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue.

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.     

Variation in nitrate metabolism in biovars of Pseudomonas solanacearum

A collection of 327 strains of Pseudomonas solanacearum , representing five biovars, was divisible into three groups on the basis of differences in nitrate metabolism. Nine strains (2.8%), of which seven were biovar 2 from bacterial wilt of potato, were nitrate reduction-deficient and failed to produce nitrite from nitrate by either of two methods of detection in five different media. A second group of 231 strains, comprising biovars 1 and 2 and a single biovar 3 strain, produced nitrite from nitrate and grew vigorously in the presence of nitrate under anaerobic conditions but were deficient in ability to denitrify. A third group comprising 57 strains of biovar 3, 28 of biovar 4 and one each of biovar 2 and 5 produced nitrite from nitrate and gave profuse growth and gas production from nitrate under anaerobic conditions. However, production of gas from nitrate (denitrification) was not a consistently reproducible property in some of the media tested. Gas production results were most reproducible when a semi-solid succinate/nitrate or glycerol/nitrate medium was used. Serial passage of four nitrate reduction-deficient isolates in nitrate medium did not restore ability to reduce nitrate.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

Citrus limonoid effects on Colorado potato beetle larval survival and development

The effects of citrus limonoids, applied topically to potato (Solanum tuberosum L. var. Katahdin) foliage, on Colorado potato beetle (Leptinotarsa decemlineata) (Say) (Chrysomelidae) larval development, growth, and survival were quantified in laboratory assays and a small-plot field test. In laboratory assays, survival, development rate, and body weight decreased with increasing limonoid concentration, however these measures of larval response did not significantly differ among varying periods of limonoid exposure (three, six, or nine days). Significant limonoid application concentration and frequency effects on survival, development rate, and defoliation were observed in the field test. These results indicate the potential utility of lethal and non-lethal effects of citrus limonoids for management of the Colorado potato beetle.     

Influence of glycosylation inhibitors on dihydropyridine binding to cardiac cells

In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v).Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-³H]PN 200-110 and v. The most severe but reversible effect occurs at 6 g/ml tunicamycin (B_max 45% and v 6%, resp., of control), a slight increase in B_max at 0.1–0.5 mM castanospermine and 0.05–2.5 mM deoxymannojirimycin. The other inhibitors gave no significant alterations of B_max.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Tumor suppression in Drosophila is causally related to the function of the lethal(2)tumorous imaginal discs gene,a dnaJ homolog

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) causes in homozygotes malignant growth of cells of the imaginal discs and the death of the mutant larvae at the time of puparium formation. We describe the molecular cloning of the 1(2)tid+ gene and its temporal expression pattern in the wild-type and mutant alleles. Germ line rescue of the tumor phenotype was achieved with a 7.0 kb Hindlll-fragment derived from the polytene chromosome band 59F5. The l(2)tid+ gene spans approximately 2.5 kb of genomic DNA. The protein coding region, 1,696 bps long, is divided by an intron into two exons. The predicted Tid⁵⁶ protein contains 518 amino acids and possesses a theoretical molecular weight of 56 kDa. It shows significant homology to all known DnaJ related proteins from bacteria, yeast, and man. The possible function of the Tid⁵⁶ protein in tumor suppression is delineated. © 1995 Wiley-Liss, Inc.